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Sample records for exon p3a due

  1. Hereditary vitamin D resistant rickets due to deletion of exon 3 of the vitamin D receptor

    SciTech Connect

    Rut, A.R.; O`Riordan, J.L.H.; Hughes, M.R.

    1994-09-01

    Hereditary vitamin D resistant rickets is an autosomal recessive disorder characterized by severe rickets, hypolcalcaemia, secondary hyperparathyroidism and occasionally, the absence of body hair. The pathological process involves resistance of target tissues to the actions of calcitriol [1,25(OH{sub 2}D{sub 3})], the hormonal form of vitamin D. Calcitriol mediates its actions through a nuclear receptor (VDR) which has been cloned and shown to be a member of the superfamily of steriod/thyroid/retinoic acid receptors. Skin fibroblasts were obtained from a Greek child with characteristic features of the condition. Total RNA was extracted from rapidly dividing cells and reverse transcribed. The coding region was amplified by PCR with primers 31a in the 5{prime} untranslated region and 31b in the 3{prime} untranslated region of the VDR cDNA sequence. The 5{prime} and 3{prime} halves of VDR were further amplified using primers tagged with M13 forward and reverse primer sequences. The whole process was carried out in duplicate starting with RNA. Sequence data was obtained using Taq dye primer cycle sequencing (ABI). Agarose gel electrophoresis revealed that the 5{prime} product was approximately 100 bp shorter than control. This was confirmed by sequencing which demonstrated a 131 bp deletion of the C-terminal part of the DNA binding domain (bases 147-277). Bases 147-277 are coded for by exon 3 and this deletion is bounded by the splice junctions. This is the first report of a deletion in VDR in any patient with vitamin D-resistant rickets. Such a deletion not only removes the second zinc finger but also results in a frameshift that corrupts the remainder of the receptor. Such a deletion may have arisen as a result of a microdeletion of genomic DNA or, more likely, as a result of defective splicing.

  2. Progression of neuropsychiatric and cognitive features due to exons 2 to 5 deletion in the epsilon-sarcoglycan gene: a case report.

    PubMed

    Multani, Namita; Moro, Elena; Lang, Anthony; Zurowski, Mateusz; Duff Canning, Sarah; Tartaglia, Maria Carmela

    2016-01-01

    Physical symptoms of myoclonus dystonia due to epsilon-sarcoglycan mutations are well documented; however, the progression of neuropsychiatric and cognitive symptoms remains unclear. We present a case of a 34-year-old woman with early childhood onset of myoclonic jerks, dystonic posture and developmental delay due to exons 2 to 5 deletion in the epsilon-sarcoglycan gene. Over time, she developed neuropsychiatric symptoms. She underwent bilateral deep brain stimulation of the ventral intermediate nucleus of the thalamus for her motor symptoms, which greatly improved but she exhibited slow deterioration of her neuropsychiatric and cognitive symptoms, particularly apathy, aggression and severe executive dysfunction. PMID:26652670

  3. Infertility due to congenital absence of vas deferens in mainly caused by variable exon 9 skipping of the CFTR gene in heterozygous males for cystic fibrosis mutations

    SciTech Connect

    Chillon, M.; Casals, T.; Nunes, V.

    1994-09-01

    About 65% or the individuals with congenital bilateral absence of the vas deferens (CBAVD) have mutations in at least one of the CFTR alleles. We have studied the phenotypic effects of the CFTR gene intron 8 polyT tract 5T allele in 90 CBAVD subjects and in parents of CF patients. This group was compared with normal individuals, and with fathers and mothers of CF patients. Allele 5T was significantly associated with CBAVD (19.6%) when compared to the general population (5.2%) ({chi}{sup 2} = 33.3%; p<<0.0001). It was represented poorly in fathers of CF patients (1.3%). Mutations were identified in one (60%) or both CFTR alleles (8.9%) of CBAVD patients. Heterozygosity for the 5T allele was strongly associated with heterozygosity for CF mutations ({chi}{sup 2} = 10.9; p<0.0004). The strong correlation between allele 5T and CBAVD, together with the low frequency of this allele in fathers of CF patients, demonstrates that variable {Delta}exon 9 produces infertility in males if associated with a CF mutation on the other chromosome. The 30% of CBAVD cases with only one CFTR mutation and without a 5T-allele may be due to other molecular mechanisms involving CFTR, distinct from {Delta}exon 9. Since there is a relatively high proportion of CBAVD without CF mutations (25%), other gene(s), distinct from CFTR, may have a role in the CBAVD phenotype.

  4. Phenotypic Variability Due to a Novel Glu292Lys Variation in Exon 8 of the BEST1 Gene Causing Best Macular Dystrophy

    PubMed Central

    Sohn, Elliott H.; Francis, Peter J.; Duncan, Jacque L.; Weleber, Richard G.; Saperstein, David A.; Farrell, Donald F.; Stone, Edwin M.

    2009-01-01

    Objective To study the phenotypic characteristics of patients with a novel p.E292K mutation in BEST1. Methods Affected individuals underwent ophthalmic examination and testing that included photography, autofluorescence, OCT, and electrophysiological testing. DNA was analyzed for BEST1 mutations. Results Five patients (aged 5–59) expressing the p.E292K mutation in BEST1 were ascertained from three families. EOG light-rise was subnormal in all probands and carriers. Carriers had normal fundus examination, mfERG, visual acuity, and were emmetropic or myopic. Only probands had hyperopia and fundus findings typical of Best macular dystrophy. OCT of vitelliform lesions demonstrated RPE elevation without subretinal fluid; atrophic lesions exhibited disruption of the hyper-reflective outer retina-RPE complex. Intense hyperautofluorescence correlated to the vitelliform lesion. Conclusions Patients with Glu292Lys variation in BEST1 exhibit intra- and interfamilial phenotypic variability. A disproportionate fraction (26%) of Best-disease-causing mutations occur in exon 8, suggesting that the portion of protein encoded by this exon (amino acids 290–316) may be especially important to bestrophin’s function. Relatively good visual acuity with vitelliform lesions can be explained by preservation of the outer retina demonstrated by OCT. Clinical relevance We demonstrate findings that can be seen with novel mutation in this region of BEST1 that carries implications for disease pathogenesis. PMID:19597114

  5. Somatic mutational analysis of FAK in breast cancer: A novel gain-of-function mutation due to deletion of exon 33

    SciTech Connect

    Fang, Xu-Qian; Liu, Xiang-Fan; Yao, Ling; Chen, Chang-Qiang; Gu, Zhi-Dong; Ni, Pei-Hua; Zheng, Xin-Min; Fan, Qi-Shi

    2014-01-10

    Highlights: •A novel FAK splicing mutation identified in breast tumor. •FAK-Del33 mutation promotes cell migration and invasion. •FAK-Del33 mutation regulates FAK/Src signal pathway. -- Abstract: Focal adhesion kinase (FAK) regulates cell adhesion, migration, proliferation, and survival. We identified a novel splicing mutant, FAK-Del33 (exon 33 deletion, KF437463), in both breast and thyroid cancers through colony sequencing. Considering the low proportion of mutant transcripts in samples, this mutation was detected by TaqMan-MGB probes based qPCR. In total, three in 21 paired breast tissues were identified with the FAK-Del33 mutation, and no mutations were found in the corresponding normal tissues. When introduced into a breast cell line through lentivirus infection, FAK-Del33 regulated cell motility and migration based on a wound healing assay. We demonstrated that the expression of Tyr397 (main auto-phosphorylation of FAK) was strongly increased in FAK-Del33 overexpressed breast tumor cells compared to wild-type following FAK/Src RTK signaling activation. These results suggest a novel and unique role of the FAK-Del33 mutation in FAK/Src signaling in breast cancer with significant implications for metastatic potential.

  6. Characterization of an Equine α-S2-Casein Variant Due to a 1.3 kb Deletion Spanning Two Coding Exons

    PubMed Central

    Brinkmann, Julia; Koudelka, Tomas; Keppler, Julia K.; Tholey, Andreas; Schwarz, Karin; Thaller, Georg; Tetens, Jens

    2015-01-01

    The production and consumption of mare’s milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows’ milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage. PMID:26444874

  7. Characterization of an Equine α-S2-Casein Variant Due to a 1.3 kb Deletion Spanning Two Coding Exons.

    PubMed

    Brinkmann, Julia; Koudelka, Tomas; Keppler, Julia K; Tholey, Andreas; Schwarz, Karin; Thaller, Georg; Tetens, Jens

    2015-01-01

    The production and consumption of mare's milk in Europe has gained importance, mainly based on positive health effects and a lower allergenic potential as compared to cows' milk. The allergenicity of milk is to a certain extent affected by different genetic variants. In classical dairy species, much research has been conducted into the genetic variability of milk proteins, but the knowledge in horses is scarce. Here, we characterize two major forms of equine αS2-casein arising from genomic 1.3 kb in-frame deletion involving two coding exons, one of which represents an equid specific duplication. Findings at the DNA-level have been verified by cDNA sequencing from horse milk of mares with different genotypes. At the protein-level, we were able to show by SDS-page and in-gel digestion with subsequent LC-MS analysis that both proteins are actually expressed. The comparison with published sequences of other equids revealed that the deletion has probably occurred before the ancestor of present-day asses and zebras diverged from the horse lineage. PMID:26444874

  8. Evolution of cyclin B3 shows an abrupt three-fold size increase, due to the extension of a single exon in placental mammals, allowing for new protein-protein interactions.

    PubMed

    Lozano, Jean-Claude; Vergé, Valérie; Schatt, Philippe; Juengel, Jennifer L; Peaucellier, Gérard

    2012-12-01

    Cyclin B3 evolution has the unique peculiarity of an abrupt 3-fold increase of the protein size in the mammalian lineage due to the extension of a single exon. We have analyzed the evolution of the gene to define the modalities of this event and the possible consequences on the function of the protein. Database searches can trace the appearance of the gene to the origin of metazoans. Most introns were already present in early metazoans, and the intron-exon structure as well as the protein size were fairly conserved in invertebrates and nonmammalian vertebrates. Although intron gains are considered as rare events, we identified two cases, one at the prochordate-chordate transition and one in murids, resulting from different mechanisms. At the emergence of mammals, the gene was relocated from chromosome 6 of platypus to the X chromosome in marsupials, but the exon extension occurred only in placental mammals. A repetitive structure of 18 amino acids, of uncertain origin, is detectable in the 3,000-nt mammalian exon-encoded sequence, suggesting an extension by multiple internal duplications, some of which are still detectable in the primate lineage. Structure prediction programs suggest that the repetitive structure has no associated three-dimensional structure but rather a tendency for disorder. Splice variant isoforms were detected in several mammalian species but without conserved pattern, notably excluding the constant coexistence of premammalian-like transcripts, without the extension. The yeast two-hybrid method revealed that, in human, the extension allowed new interactions with ten unrelated proteins, most of them with specific three-dimensional structures involved in protein-protein interactions, and some highly expressed in testis, as is cyclin B3. The interactions with activator of cAMP-responsive element modulator in testis (ACT), germ cell-less homolog 1, and chromosome 1 open reading frame 14 remain to be verified in vivo since they may not be expressed

  9. Inhomogeneous DNA: Conducting exons and insulating introns

    NASA Astrophysics Data System (ADS)

    Krokhin, A. A.; Bagci, V. M. K.; Izrailev, F. M.; Usatenko, O. V.; Yampol'Skii, V. A.

    2009-08-01

    Parts of DNA sequences known as exons and introns play very different roles in coding and storage of genetic information. Here we show that their conducting properties are also very different. Taking into account long-range correlations among four basic nucleotides that form double-stranded DNA sequence, we calculate electron localization length for exon and intron regions. Analyzing different DNA molecules, we obtain that the exons have narrow bands of extended states, unlike the introns where all the states are well localized. The band of extended states is due to a specific form of the binary correlation function of the sequence of basic DNA nucleotides.

  10. In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

    PubMed Central

    Sterner, D A; Berget, S M

    1993-01-01

    Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron. Images PMID:7682652

  11. Genomic features defining exonic variants that modulate splicing

    PubMed Central

    2010-01-01

    Background Single point mutations at both synonymous and non-synonymous positions within exons can have severe effects on gene function through disruption of splicing. Predicting these mutations in silico purely from the genomic sequence is difficult due to an incomplete understanding of the multiple factors that may be responsible. In addition, little is known about which computational prediction approaches, such as those involving exonic splicing enhancers and exonic splicing silencers, are most informative. Results We assessed the features of single-nucleotide genomic variants verified to cause exon skipping and compared them to a large set of coding SNPs common in the human population, which are likely to have no effect on splicing. Our findings implicate a number of features important for their ability to discriminate splice-affecting variants, including the naturally occurring density of exonic splicing enhancers and exonic splicing silencers of the exon and intronic environment, extensive changes in the number of predicted exonic splicing enhancers and exonic splicing silencers, proximity to the splice junctions and evolutionary constraint of the region surrounding the variant. By extending this approach to additional datasets, we also identified relevant features of variants that cause increased exon inclusion and ectopic splice site activation. Conclusions We identified a number of features that have statistically significant representation among exonic variants that modulate splicing. These analyses highlight putative mechanisms responsible for splicing outcome and emphasize the role of features important for exon definition. We developed a web-tool, Skippy, to score coding variants for these relevant splice-modulating features. PMID:20158892

  12. Updating P300: An Integrative Theory of P3a and P3b

    PubMed Central

    Polich, John

    2009-01-01

    The empirical and theoretical development of the P300 event-related brain potential (ERP) is reviewed by considering factors that contribute to its amplitude, latency, and general characteristics. The neuropsychological origins of the P3a and P3b subcomponents are detailed, and how target/standard discrimination difficulty modulates scalp topography is discussed. The neural loci of P3a and P3b generation are outlined, and a cognitive model is proffered: P3a originates from stimulus-driven frontal attention mechanisms during task processing, whereas P3b originates from temporal-parietal activity associated with attention and appears related to subsequent memory processing. Neurotransmitter actions associating P3a to frontal/dopaminergic and P3b to parietal/norepinephrine pathways are highlighted. Neuroinhibition is suggested as an overarching theoretical mechanism for P300, which is elicited when stimulus detection engages memory operations. PMID:17573239

  13. The evolution of the coding exome of the Arabidopsis species - the influences of DNA methylation, relative exon position, and exon length

    PubMed Central

    2014-01-01

    Background The evolution of the coding exome is a major driving force of functional divergence both between species and between protein isoforms. Exons at different positions in the transcript or in different transcript isoforms may (1) mutate at different rates due to variations in DNA methylation level; and (2) serve distinct biological roles, and thus be differentially targeted by natural selection. Furthermore, intrinsic exonic features, such as exon length, may also affect the evolution of individual exons. Importantly, the evolutionary effects of these intrinsic/extrinsic features may differ significantly between animals and plants. Such inter-lineage differences, however, have not been systematically examined. Results Here we examine how DNA methylation at CpG dinucleotides (CpG methylation), in the context of intrinsic exonic features (exon length and relative exon position in the transcript), influences the evolution of coding exons of Arabidopsis thaliana. We observed fairly different evolutionary patterns in A. thaliana as compared with those reported for animals. Firstly, the mutagenic effect of CpG methylation is the strongest for internal exons and the weakest for first exons despite the stringent selective constraints on the former group. Secondly, the mutagenic effect of CpG methylation increases significantly with length in first exons but not in the other two exon groups. Thirdly, CpG methylation level is correlated with evolutionary rates (dS, dN, and the dN/dS ratio) with markedly different patterns among the three exon groups. The correlations are generally positive, negative, and mixed for first, last, and internal exons, respectively. Fourthly, exon length is a CpG methylation-independent indicator of evolutionary rates, particularly for dN and the dN/dS ratio in last and internal exons. Finally, the evolutionary patterns of coding exons with regard to CpG methylation differ significantly between Arabidopsis species and mammals. Conclusions

  14. Severe von Willebrand disease due to a defect at the level of von Willebrand factor mRNA expression: Detection by exonic PCR-restriction fragment length polymorphism analysis

    SciTech Connect

    Nichols, W.C.; Lyons, S.E.; Harrison, J.S.; Cody, R.L.; Ginsburg, D. )

    1991-05-01

    von Willebrand disease (vWD), the most common inherited bleeding disorder in humans, results from abnormalities in the plasma clotting protein von Willebrand factor (vWF). Severe (type III) vWD is autosomal recessive in inheritance and is associated with extremely low or undetectable vWF levels. The authors report a method designed to distinguish mRNA expression from the two vWF alleles by PCR analysis of peripheral blood platelet RNA using DNA sequence polymorphisms located within exons of the vWF gene. This approach was applied to a severe-vWD pedigree in which three of eight siblings are affected and the parents and additional siblings are clinically normal. Each parent was shown to carry a vWF allele that is silent at the mRNA level. Family members inheriting both abnormal alleles are affected with severe vWD, whereas individuals with only one abnormal allele are asymptomatic. Given the frequencies of the two exon polymorphisms reported here, this analysis should be applicable to {approx}70% of type I and type III vWD patients. This comparative DNA and RNA PCR-restriction fragment length polymorphism approach may also prove useful in identifying defects at the level of gene expression associated with other genetic disorders.

  15. Distinct pattern of P3a event-related potential in borderline personality disorder.

    PubMed

    Meares, Russell; Melkonian, Dmitriy; Gordon, Evian; Williams, Leanne

    2005-02-28

    P3a and P3b event-related brain potentials to auditory stimuli were recorded for 17 unmedicated patients with borderline personality disorder, 17 matched healthy controls and 100 healthy control participants spanning five decades. Using high-resolution fragmentary decomposition for single-trial event-related potential analysis, distinctive disturbances in P3a in borderline personality disorder patients were found: abnormally enhanced amplitude, failure to habituate and a loss of temporal locking with P3b. Normative age dependencies from 100 controls suggest that natural age-related decline in P3a amplitude is reduced in borderline personality disorder patients and is likely to indicate failure of frontal maturation. On the basis of the theories of Hughlings Jackson, this conceptualization of borderline personality disorder is consistent with an aetiological model of borderline personality disorder. PMID:15706238

  16. Exons, Introns, and DNA Thermodynamics

    NASA Astrophysics Data System (ADS)

    Carlon, Enrico; Malki, Mehdi Lejard; Blossey, Ralf

    2005-05-01

    The genes of eukaryotes are characterized by protein coding fragments, the exons, interrupted by introns, i.e., stretches of DNA which do not carry useful information for protein synthesis. We have analyzed the melting behavior of randomly selected human cDNA sequences obtained from genomic DNA by removing all introns. A clear correspondence is observed between exons and melting domains. This finding may provide new insights into the physical mechanisms underlying the evolution of genes.

  17. Meditation (Vipassana) and the P3a Event-Related Brain Potential

    PubMed Central

    Cahn, B. Rael; Polich, John

    2009-01-01

    A three-stimulus auditory oddball series was presented to experienced Vipassana meditators during meditation and a control thought period to elicit event-related brain potentials (ERPs) in the two different mental states. The stimuli consisted of a frequent standard tone (500 Hz), an infrequent oddball tone (1000 Hz), and an infrequent distracter (white noise), with all stimuli passively presented through headphones and no task imposed. The strongest meditation compared to control state effects occurred for the distracter stimuli: N1 amplitude from the distracter was reduced frontally during meditation; P2 amplitude from both the distracter and oddball stimuli were somewhat reduced during meditation; P3a amplitude from the distracter was reduced during meditation. The meditation-induced reduction in P3a amplitude was strongest in participants reporting more hours of daily meditation practice and was not evident in participants reporting drowsiness during their experimental meditative session. The findings suggest that meditation state can decrease the amplitude of neurophysiologic processes that subserve attentional engagement elicited by unexpected and distracting stimuli. Consistent with the aim of Vipassana meditation to reduce cognitive and emotional reactivity, the state effect of reduced P3a amplitude to distracting stimuli reflects decreased automated reactivity and evaluative processing of task irrelevant attention-demanding stimuli. PMID:18845193

  18. Neural generators of the auditory evoked potential components P3a and P3b.

    PubMed

    Wronka, Eligiusz; Kaiser, Jan; Coenen, Anton M L

    2012-01-01

    The aim of the present study was to define the scalp topography of the two subcomponents of the P3 component of the auditory evoked potential elicited in a three-stimulus oddball paradigm and to identify their cortical generators using the standardized low resolution electromagnetic tomography (sLORETA). Subjects were presented with a random sequence of auditory stimuli and instructed to respond to an infrequently occurring target stimulus inserted into a sequence of frequent standard and rare non-target stimuli. Results show that the magnitude of the frontal P3a is determined by the relative physical difference among stimuli, as it was larger for the stimulus more deviant from the standard. Major neural generators of the P3a were localized within frontal cortex and anterior cingulate gyrus. In contrast to this, the P3b, showing maximal amplitude at parietal locations, was larger for stimuli demanding a response than for the rare non-target. Major sources of the P3b included the superior parietal lobule and the posterior part of the cingulate gyrus. Our findings are in line with the hypothesis that P3a is related to alerting activity during the initial allocation of attention, while P3b is related to activation of a posterior network when the neuronal model of perceived stimulation is compared with the attentional trace. PMID:22508084

  19. Overview on DMD exon skipping.

    PubMed

    Aartsma-Rus, Annemieke

    2012-01-01

    Antisense-mediated exon skipping to restore the disrupted dystrophin reading frame is currently in clinical trials for Duchenne muscular dystrophy. This chapter describes the rationale of this approach and gives an overview of in vitro and in vivo experiments with antisense oligonucleotides and antisense genes. Finally, an overview of clinical trials is given and outstanding questions and hurdles are discussed. PMID:22454057

  20. Antisense-mediated exon inclusion

    PubMed Central

    Hua, Yimin; Krainer, Adrian R

    2012-01-01

    Exon skipping induced by gene mutations is a common mechanism responsible for many genetic diseases. A practical approach to correct the aberrant splicing of defective genes is to use antisense oligonucleotides (ASOs). The recognition of splice sites and the regulation of splicing involve multiple positive or negative cis-acting elements and trans-acting factors. Base-pairing of ASOs to a negative element in a targeted pre-mRNA blocks the binding of splicing repressors to this cis-element and/or disrupts an unfavorable secondary structure; as a result, the ASO restores exon inclusion. For example, we have recently shown that appropriate 2’-O-(2-methoxyethyl) (MOE) phosphorothioate-modified ASOs can efficiently correct survival motor neuron 2 (SMN2) exon 7 splicing in a cell-free splicing assay, in cultured human cells—including patient fibroblasts—and in both peripheral tissues and the CNS of SMA mouse models. These ASOs are promising drug leads for SMA therapy. PMID:22454070

  1. Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

    PubMed Central

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-01-01

    The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. PMID:9410897

  2. Disruption of the splicing enhancer sequence within exon 27 of the dystrophin gene by a nonsense mutation induces partial skipping of the exon and is responsible for Becker muscular dystrophy.

    PubMed

    Shiga, N; Takeshima, Y; Sakamoto, H; Inoue, K; Yokota, Y; Yokoyama, M; Matsuo, M

    1997-11-01

    The mechanism of exon skipping induced by nonsense mutations has not been well elucidated. We now report results of in vitro splicing studies which disclosed that a particular example of exon skipping is due to disruption of a splicing enhancer sequence located within the exon. A nonsense mutation (E1211X) due to a G to T transversion at the 28th nucleotide of exon 27 (G3839T) was identified in the dystrophin gene of a Japanese Becker muscular dystrophy case. Partial skipping of the exon resulted in the production of truncated dystrophin mRNA, although the consensus sequences for splicing at both ends of exon 27 were unaltered. To determine how E1211X induced exon 27 skipping, the splicing enhancer activity of purine-rich region within exon 27 was examined in an in vitro splicing system using chimeric doublesex gene pre-mRNA. The mutant sequence containing G3839T abolished splicing enhancer activity of the wild-type purine-rich sequence for the upstream intron in this chimeric pre-mRNA. An artificial polypurine oligonucleotide mimicking the purine-rich sequence of exon 27 also showed enhancer activity that was suppressed by the introduction of a T nucleotide. Furthermore, the splicing enhancer activity was more markedly inhibited when a nonsense codon was created by the inserted T residue. This is the first evidence that partial skipping of an exon harboring a nonsense mutation is due to disruption of a splicing enhancer sequence. PMID:9410897

  3. Pitfalls in prenatal diagnosis of DMD due to placental mosaicism of the X-chromosomes: prenatal and postnatal findings in a fetus with a deletion of exons 67-71 of the dystrophin gene.

    PubMed

    Vondran, S; Edelmann, J; Holland, H; Wolf, C; Strenge, S; Thamm, B; Thiele, H; Froster, U G

    1999-01-01

    Prenatal diagnosis of Duchenne and Becker muscular dystrophy (DMD) is performed as a routine procedure in many laboratories. The major potential problem is an incorrect diagnosis that could be obtained due to contamination with maternal tissue. We report a case of mosaicism of the X-chromosomes confined to the placenta as a possible source of confusing results in prenatal diagnosis of DMD. To the best of our knowledge, this is the first reported case of this problem in a prenatal DMD diagnosis. PMID:10073911

  4. Identification and characterization of alternative exon usage linked glioblastoma multiforme survival

    PubMed Central

    2012-01-01

    Background Alternative exon usage (AEU) is an important component of gene regulation. Exon expression platforms allow the detection of associations between AEU and phenotypes such as cancer. Numerous studies have identified associations between gene expression and the brain cancer glioblastoma multiforme (GBM). The few consistent gene expression biomarkers of GBM that have been reported may be due to the limited consideration of AEU and the analytical approaches used. The objectives of this study were to develop a model that accounts for the variations in expression present between the exons within a gene and to identify AEU biomarkers of GBM survival. Methods The expression of exons corresponding to 25,403 genes was related to the survival of 250 individuals diagnosed with GBM in a training data set. Genes exhibiting AEU in the training data set were confirmed in an independent validation data set of 78 patients. A hierarchical mixed model that allows the consideration of covariation between exons within a gene and of the effect of the epidemiological characteristics of the patients was developed to identify associations between exon expression and patient survival. This general model describes all three possible scenarios: multi-exon genes with and without AEU, and single-exon genes. Results AEU associated with GBM survival was identified on 2477 genes (P-value < 5.0E-04 or FDR-adjusted P-value < 0.05). G-protein coupled receptor 98 (Gpr98) and epidermal growth factor (Egf) were among the genes exhibiting AEU with 30 and 9 exons associated with GBM survival, respectively. Pathways enriched among the AEU genes included focal adhesion, ECM-receptor interaction, ABC transporters and pathways in cancer. In addition, 24 multi-exon genes without AEU and 8 single-exon genes were associated with GBM survival (FDR-adjusted P-value < 0.05). Conclusions The inferred patterns of AEU were consistent with in silico AS models. The hierarchical model used offered a flexible and

  5. High Resolution Melting Analysis for JAK2 Exon 14 and Exon 12 Mutations

    PubMed Central

    Rapado, Inmaculada; Grande, Silvia; Albizua, Enriqueta; Ayala, Rosa; Hernández, José-Angel; Gallardo, Miguel; Gilsanz, Florinda; Martinez-Lopez, Joaquin

    2009-01-01

    JAK2 mutations are important criteria for the diagnosis of Philadelphia chromosome-negative myeloproliferative neoplasms. We aimed to assess JAK2 exon 14 and exon 12 mutations by high-resolution melting (HRM) analysis, which allows variation screening. The exon 14 analysis included 163 patients with polycythemia vera, secondary erythrocytoses, essential thrombocythemia, or secondary thrombocytoses, and 126 healthy subjects. The study of exon 12 included 40 JAK2 V617F-negative patients (nine of which had polycythemia vera, and 31 with splanchnic vein thrombosis) and 30 healthy subjects. HRM analyses of JAK2 exons 14 and 12 gave analytical sensitivities near 1% and both intra- and interday coefficients of variation of less than 1%. For HRM analysis of JAK2 exon 14 in polycythemia vera and essential thrombocythemia, clinical sensitivities were 93.5% and 67.9%, clinical specificities were 98.8% and 97.0%, positive predictive values were 93.5% and 79.2%, and negative predictive values were 98.8% and 94.6, respectively. Correlations were observed between the results from HRM and three commonly used analytical methods. The JAK2 exon 12 HRM results agreed completely with those from sequencing analysis, and the three mutations in exon 12 were detected by both methods. Hence, HRM analysis of exons 14 and 12 in JAK2 shows better diagnostic values than three other routinely used methods against which it was compared. In addition, HRM analysis has the advantage of detecting unknown mutations. PMID:19225136

  6. Purifying Selection on Exonic Splice Enhancers in Intronless Genes.

    PubMed

    Savisaar, Rosina; Hurst, Laurence D

    2016-06-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  7. Targeted Exon Sequencing in Usher Syndrome Type I

    PubMed Central

    Bujakowska, Kinga M.; Consugar, Mark; Place, Emily; Harper, Shyana; Lena, Jaclyn; Taub, Daniel G.; White, Joseph; Navarro-Gomez, Daniel; Weigel DiFranco, Carol; Farkas, Michael H.; Gai, Xiaowu; Berson, Eliot L.; Pierce, Eric A.

    2014-01-01

    Purpose. Patients with Usher syndrome type I (USH1) have retinitis pigmentosa, profound congenital hearing loss, and vestibular ataxia. This syndrome is currently thought to be associated with at least six genes, which are encoded by over 180 exons. Here, we present the use of state-of-the-art techniques in the molecular diagnosis of a cohort of 47 USH1 probands. Methods. The cohort was studied with selective exon capture and next-generation sequencing of currently known inherited retinal degeneration genes, comparative genomic hybridization, and Sanger sequencing of new USH1 exons identified by human retinal transcriptome analysis. Results. With this approach, we were able to genetically solve 14 of the 47 probands by confirming the biallelic inheritance of mutations. We detected two likely pathogenic variants in an additional 19 patients, for whom family members were not available for cosegregation analysis to confirm biallelic inheritance. Ten patients, in addition to primary disease–causing mutations, carried rare likely pathogenic USH1 alleles or variants in other genes associated with deaf-blindness, which may influence disease phenotype. Twenty-one of the identified mutations were novel among the 33 definite or likely solved patients. Here, we also present a clinical description of the studied cohort at their initial visits. Conclusions. We found a remarkable genetic heterogeneity in the studied USH1 cohort with multiplicity of mutations, of which many were novel. No obvious influence of genotype on phenotype was found, possibly due to small sample sizes of the genotypes under study. PMID:25468891

  8. An Exon-Capture System for the Entire Class Ophiuroidea

    PubMed Central

    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. PMID:26474846

  9. An Exon-Capture System for the Entire Class Ophiuroidea.

    PubMed

    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. PMID:26474846

  10. Purifying Selection on Exonic Splice Enhancers in Intronless Genes

    PubMed Central

    Savisaar, Rosina; Hurst, Laurence D.

    2016-01-01

    Exonic splice enhancers (ESEs) are short nucleotide motifs, enriched near exon ends, that enhance the recognition of the splice site and thus promote splicing. Are intronless genes under selection to avoid these motifs so as not to attract the splicing machinery to an mRNA that should not be spliced, thereby preventing the production of an aberrant transcript? Consistent with this possibility, we find that ESEs in putative recent retrocopies are at a higher density and evolving faster than those in other intronless genes, suggesting that they are being lost. Moreover, intronless genes are less dense in putative ESEs than intron-containing ones. However, this latter difference is likely due to the skewed base composition of intronless sequences, a skew that is in line with the general GC richness of few exon genes. Indeed, after controlling for such biases, we find that both intronless and intron-containing genes are denser in ESEs than expected by chance. Importantly, nucleotide-controlled analysis of evolutionary rates at synonymous sites in ESEs indicates that the ESEs in intronless genes are under purifying selection in both human and mouse. We conclude that on the loss of introns, some but not all, ESE motifs are lost, the remainder having functions beyond a role in splice promotion. These results have implications for the design of intronless transgenes and for understanding the causes of selection on synonymous sites. PMID:26802218

  11. Absolute pitch: evidence for early cognitive facilitation during passive listening as revealed by reduced P3a amplitudes.

    PubMed

    Rogenmoser, Lars; Elmer, Stefan; Jäncke, Lutz

    2015-03-01

    Absolute pitch (AP) is the rare ability to identify or produce different pitches without using reference tones. At least two sequential processing stages are assumed to contribute to this phenomenon. The first recruits a pitch memory mechanism at an early stage of auditory processing, whereas the second is driven by a later cognitive mechanism (pitch labeling). Several investigations have used active tasks, but it is unclear how these two mechanisms contribute to AP during passive listening. The present work investigated the temporal dynamics of tone processing in AP and non-AP (NAP) participants by using EEG. We applied a passive oddball paradigm with between- and within-tone category manipulations and analyzed the MMN reflecting the early stage of auditory processing and the P3a response reflecting the later cognitive mechanism during the second processing stage. Results did not reveal between-group differences in MMN waveforms. By contrast, the P3a response was specifically associated with AP and sensitive to the processing of different pitch types. Specifically, AP participants exhibited smaller P3a amplitudes, especially in between-tone category conditions, and P3a responses correlated significantly with the age of commencement of musical training, suggesting an influence of early musical exposure on AP. Our results reinforce the current opinion that the representation of pitches at the processing level of the auditory-related cortex is comparable among AP and NAP participants, whereas the later processing stage is critical for AP. Results are interpreted as reflecting cognitive facilitation in AP participants, possibly driven by the availability of multiple codes for tones. PMID:25170790

  12. MET Exon 14 Alterations in Lung Cancer: Exon Skipping Extends Half-Life.

    PubMed

    Drilon, Alexander

    2016-06-15

    MET exon 14 alterations are a diverse group of mutations, many of which disrupt splice acceptor or donor sites leading to exon 14 skipping, impaired receptor degradation, and oncogenic transformation. These alterations are clinically targetable with MET-directed therapy. Clin Cancer Res; 22(12); 2832-4. ©2016 AACRSee related article by Tong et al., p. 3048. PMID:27009743

  13. Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression

    PubMed Central

    Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M.; Miller, Tyler E.; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.; Bredel, Markus

    2014-01-01

    Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

  14. Lineage-specific splicing of a brain-enriched alternative exon promotes glioblastoma progression.

    PubMed

    Ferrarese, Roberto; Harsh, Griffith R; Yadav, Ajay K; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Dombrowski, Stephen M; Miller, Tyler E; Masilamani, Anie P; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M; Yu, Irene L Y; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; von Elverfeldt, Dominik; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W; He, Xiaolin; Prinz, Marco; Chandler, James P; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N; Carro, Maria S; Bredel, Markus

    2014-07-01

    Tissue-specific alternative splicing is critical for the emergence of tissue identity during development, yet the role of this process in malignant transformation is undefined. Tissue-specific splicing involves evolutionarily conserved, alternative exons that represent only a minority of the total alternative exons identified. Many of these conserved exons have functional features that influence signaling pathways to profound biological effect. Here, we determined that lineage-specific splicing of a brain-enriched cassette exon in the membrane-binding tumor suppressor annexin A7 (ANXA7) diminishes endosomal targeting of the EGFR oncoprotein, consequently enhancing EGFR signaling during brain tumor progression. ANXA7 exon splicing was mediated by the ribonucleoprotein PTBP1, which is normally repressed during neuronal development. PTBP1 was highly expressed in glioblastomas due to loss of a brain-enriched microRNA (miR-124) and to PTBP1 amplification. The alternative ANXA7 splicing trait was present in precursor cells, suggesting that glioblastoma cells inherit the trait from a potential tumor-initiating ancestor and that these cells exploit this trait through accumulation of mutations that enhance EGFR signaling. Our data illustrate that lineage-specific splicing of a tissue-regulated alternative exon in a constituent of an oncogenic pathway eliminates tumor suppressor functions and promotes glioblastoma progression. This paradigm may offer a general model as to how tissue-specific regulatory mechanisms can reprogram normal developmental processes into oncogenic ones. PMID:24865424

  15. Menzerath-Altmann law in mammalian exons reflects the dynamics of gene structure evolution.

    PubMed

    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. PMID:25155263

  16. Oxidative Stress Triggers Body-Wide Skipping of Multiple Exons of the Spinal Muscular Atrophy Gene

    PubMed Central

    Seo, Joonbae; Singh, Natalia N.; Ottesen, Eric W.; Sivanesan, Senthilkumar; Shishimorova, Maria; Singh, Ravindra N.

    2016-01-01

    Humans carry two nearly identical copies of Survival Motor Neuron gene: SMN1 and SMN2. Loss of SMN1 leads to spinal muscular atrophy (SMA), the most frequent genetic cause of infant mortality. While SMN2 cannot compensate for the loss of SMN1 due to predominant skipping of exon 7, correction of SMN2 exon 7 splicing holds the promise of a cure for SMA. Previously, we used cell-based models coupled with a multi-exon-skipping detection assay (MESDA) to demonstrate the vulnerability of SMN2 exons to aberrant splicing under the conditions of oxidative stress (OS). Here we employ a transgenic mouse model and MESDA to examine the OS-induced splicing regulation of SMN2 exons. We induced OS using paraquat that is known to trigger production of reactive oxygen species and cause mitochondrial dysfunction. We show an overwhelming co-skipping of SMN2 exon 5 and exon 7 under OS in all tissues except testis. We also show that OS increases skipping of SMN2 exon 3 in all tissues except testis. We uncover several new SMN2 splice isoforms expressed at elevated levels under the conditions of OS. We analyze cis-elements and transacting factors to demonstrate the diversity of mechanisms for splicing misregulation under OS. Our results of proteome analysis reveal downregulation of hnRNP H as one of the potential consequences of OS in brain. Our findings suggest SMN2 as a sensor of OS with implications to SMA and other diseases impacted by low levels of SMN protein. PMID:27111068

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

    PubMed

    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. PMID:26320575

  18. Tau exon 10 alternative splicing and tauopathies

    PubMed Central

    Liu, Fei; Gong, Cheng-Xin

    2008-01-01

    Abnormalities of microtubule-associated protein tau play a central role in neurofibrillary degeneration in several neurodegenerative disorders that collectively called tauopathies. Six isoforms of tau are expressed in adult human brain, which result from alternative splicing of pre-mRNA generated from a single tau gene. Alternative splicing of tau exon 10 results in tau isoforms containing either three or four microtubule-binding repeats (3R-tau and 4R-tau, respectively). Approximately equal levels of 3R-tau and 4R-tau are expressed in normal adult human brain, but the 3R-tau/4R-tau ratio is altered in the brains in several tauopathies. Discovery of silence mutations and intronic mutations of tau gene in some individuals with frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), which only disrupt tau exon 10 splicing but do not alter tau's primary sequence, demonstrates that dysregulation of tau exon 10 alternative splicing and consequently of 3R-tau/4R-tau balance is sufficient to cause neurodegeneration and dementia. Here, we review the gene structure, transcripts and protein isoforms of tau, followed by the regulation of exon 10 splicing that determines the expression of 3R-tau or 4R-tau. Finally, dysregulation of exon 10 splicing of tau in several tauopathies is discussed. Understanding the molecular mechanisms by which tau exon 10 splicing is regulated and how it is disrupted in tauopathies will provide new insight into the mechanisms of these tauopathies and help identify new therapeutic targets to treat these disorders. PMID:18616804

  19. Tropomyosin exons as models for alternative splicing.

    PubMed

    Gooding, Clare; Smith, Christopher W J

    2008-01-01

    Three of the four mammalian tropomyosin (Tm) genes are alternatively spliced, most commonly by mutually exclusive selection from pairs of internal or 3' end exons. Alternative splicing events in the TPM1, 2 and 3 genes have been analysed experimentally in various levels ofdetail. In particular, mutually exclusive exon pairs in the betaTm (TPM2) and alphaTm (TPM1) genes are among the most intensively studied models for striated and smooth muscle specific alternative splicing, respectively. Analysis of these model systems has provided important insights into general mechanisms and strategies of splicing regulation. PMID:19209811

  20. Exon skipping therapy for Duchenne muscular dystrophy.

    PubMed

    Kole, Ryszard; Krieg, Arthur M

    2015-06-29

    Duchenne muscular dystrophy (DMD) is caused mostly by internal deletions in the gene for dystrophin, a protein essential for maintaining muscle cell membrane integrity. These deletions abrogate the reading frame and the lack of dystrophin results in progressive muscle deterioration. DMD patients experience progressive loss of ambulation, followed by a need for assisted ventilation, and eventual death in mid-twenties. By the method of exon skipping in dystrophin pre-mRNA the reading frame is restored and the internally deleted but functional dystrophin is produced. Two oligonucleotide drugs that induce desired exon skipping are currently in advanced clinical trials. PMID:25980936

  1. A survey on intron and exon lengths.

    PubMed Central

    Hawkins, J D

    1988-01-01

    The lengths of introns and exons in various parts of genes of vertebrates, insects, plants and fungi are tabulated. Differences between the various groups of organisms are apparent. The results are discussed and support the idea that, generally speaking, introns were present in primitive genomes, though in some cases they may have been inserted into pre-existing genes. PMID:3057449

  2. Exon Snipping in Duchenne Muscular Dystrophy.

    PubMed

    Kemaladewi, Dwi U; Cohn, Ronald D

    2016-03-01

    Duchenne muscular dystrophy (DMD) is a life-limiting neuromuscular disorder caused by mutations in the DMD gene encoding dystrophin. We discuss very recent studies that used CRISPR/Cas9 technology to 'snip out' mutated exons in DMD, restoring the reading frame of the gene. We also present cautionary aspects of translating this exciting technology into clinical practice. PMID:26856237

  3. Nucleocytoplasmic transfer of the NF2 tumor suppressor protein merlin is regulated by exon 2 and a CRM1-dependent nuclear export signal in exon 15.

    PubMed

    Kressel, Michael; Schmucker, Beatrice

    2002-09-15

    The neurofibromatosis 2 protein merlin is a classical tumor suppressor protein. Germline mutations predispose to the development of schwannomas, meningiomas and ependymomas. Merlin has been implicated in cellular migration and adhesion. This function is reflected in its subcellular localization at the plasma membrane and known interacting partners. Merlin has been regarded as an exception in not exerting a functional role within the nucleus as other tumor suppressors do. Here, we show that detection of wild-type protein in the nucleus is a rare event. However, splicing out of exon 2 leads to unrestricted entry into the nucleus. Skipping of adjacent exon 3 has no comparable effect ruling out an unspecific effect due to misfolding of the 4.1/JEF domain. Exon 2 functions as a cytoplasmic retention factor as it is able to confer sole cytoplasmic localization to a GFP fusion protein. Nuclear entry of merlin is thus regulated by alternative splicing within the 4.1/JEF domain and analogous to band 4.1 protein. Merlin's ability to enter the nucleus is complemented by a full nuclear-cytoplasmic shuttle protein with a functional Rev-type nuclear export sequence (NES) within exon 15 that facilitates export via the CRM1/exportin pathway. Deletion of this NES or treatment with the CRM1-specific inhibitor leptomycin B leads to overall nuclear accumulation of merlin isoforms missing exon 2. A cellular function different to the wild-type protein is implied for naturally occurring splice variants lacking exon 2. A putative effect of merlin as a transcriptional regulator and identification of nuclear binding partners remains to be elucidated. PMID:12217955

  4. Exonic remnants of whole-genome duplication reveal cis-regulatory function of coding exons

    PubMed Central

    Dong, Xianjun; Navratilova, Pavla; Fredman, David; Drivenes, Øyvind; Becker, Thomas S.; Lenhard, Boris

    2010-01-01

    Using a comparative genomics approach to reconstruct the fate of genomic regulatory blocks (GRBs) and identify exonic remnants that have survived the disappearance of their host genes after whole-genome duplication (WGD) in teleosts, we discover a set of 38 candidate cis-regulatory coding exons (RCEs) with predicted target genes. These elements demonstrate evolutionary separation of overlapping protein-coding and regulatory information after WGD in teleosts. We present evidence that the corresponding mammalian exons are still under both coding and non-coding selection pressure, are more conserved than other protein coding exons in the host gene and several control sets, and share key characteristics with highly conserved non-coding elements in the same regions. Their dual function is corroborated by existing experimental data. Additionally, we show examples of human exon remnants stemming from the vertebrate 2R WGD. Our findings suggest that long-range cis-regulatory inputs for developmental genes are not limited to non-coding regions, but can also overlap the coding sequence of unrelated genes. Thus, exonic regulatory elements in GRBs might be functionally equivalent to those in non-coding regions, calling for a re-evaluation of the sequence space in which to look for long-range regulatory elements and experimentally test their activity. PMID:19969543

  5. ExSurv: A Web Resource for Prognostic Analyses of Exons Across Human Cancers Using Clinical Transcriptomes.

    PubMed

    Hashemikhabir, Seyedsasan; Budak, Gungor; Janga, Sarath Chandra

    2016-01-01

    Survival analysis in biomedical sciences is generally performed by correlating the levels of cellular components with patients' clinical features as a common practice in prognostic biomarker discovery. While the common and primary focus of such analysis in cancer genomics so far has been to identify the potential prognostic genes, alternative splicing - a posttranscriptional regulatory mechanism that affects the functional form of a protein due to inclusion or exclusion of individual exons giving rise to alternative protein products, has increasingly gained attention due to the prevalence of splicing aberrations in cancer transcriptomes. Hence, uncovering the potential prognostic exons can not only help in rationally designing exon-specific therapeutics but also increase specificity toward more personalized treatment options. To address this gap and to provide a platform for rational identification of prognostic exons from cancer transcriptomes, we developed ExSurv (https://exsurv.soic.iupui.edu), a web-based platform for predicting the survival contribution of all annotated exons in the human genome using RNA sequencing-based expression profiles for cancer samples from four cancer types available from The Cancer Genome Atlas. ExSurv enables users to search for a gene of interest and shows survival probabilities for all the exons associated with a gene and found to be significant at the chosen threshold. ExSurv also includes raw expression values across the cancer cohort as well as the survival plots for prognostic exons. Our analysis of the resulting prognostic exons across four cancer types revealed that most of the survival-associated exons are unique to a cancer type with few processes such as cell adhesion, carboxylic, fatty acid metabolism, and regulation of T-cell signaling common across cancer types, possibly suggesting significant differences in the posttranscriptional regulatory pathways contributing to prognosis. PMID:27528797

  6. ExSurv: A Web Resource for Prognostic Analyses of Exons Across Human Cancers Using Clinical Transcriptomes

    PubMed Central

    Hashemikhabir, Seyedsasan; Budak, Gungor; Janga, Sarath Chandra

    2016-01-01

    Survival analysis in biomedical sciences is generally performed by correlating the levels of cellular components with patients’ clinical features as a common practice in prognostic biomarker discovery. While the common and primary focus of such analysis in cancer genomics so far has been to identify the potential prognostic genes, alternative splicing – a posttranscriptional regulatory mechanism that affects the functional form of a protein due to inclusion or exclusion of individual exons giving rise to alternative protein products, has increasingly gained attention due to the prevalence of splicing aberrations in cancer transcriptomes. Hence, uncovering the potential prognostic exons can not only help in rationally designing exon-specific therapeutics but also increase specificity toward more personalized treatment options. To address this gap and to provide a platform for rational identification of prognostic exons from cancer transcriptomes, we developed ExSurv (https://exsurv.soic.iupui.edu), a web-based platform for predicting the survival contribution of all annotated exons in the human genome using RNA sequencing-based expression profiles for cancer samples from four cancer types available from The Cancer Genome Atlas. ExSurv enables users to search for a gene of interest and shows survival probabilities for all the exons associated with a gene and found to be significant at the chosen threshold. ExSurv also includes raw expression values across the cancer cohort as well as the survival plots for prognostic exons. Our analysis of the resulting prognostic exons across four cancer types revealed that most of the survival-associated exons are unique to a cancer type with few processes such as cell adhesion, carboxylic, fatty acid metabolism, and regulation of T-cell signaling common across cancer types, possibly suggesting significant differences in the posttranscriptional regulatory pathways contributing to prognosis. PMID:27528797

  7. Association between polymorphisms of exon 12 and exon 24 of JHDM2A gene and male infertility

    PubMed Central

    Hojati, Zohreh; Nouri Emamzadeh, Fatemeh; Dehghanian, Fariba

    2016-01-01

    Background: Some dynamic changes occurs during spermatogenesis such as histone removal and its replacement with transition nuclear protein and protamine. These proteins are required for packing and condensation of sperm chromatin. JHDM2A is a histone demethylase that directly binds to promoter regions of Tnp1 and Prm1 genes and controls their expression by removing H3K9 at their promoters. Objective: The association between polymorphisms of exon 12 and exon 24 in JHDM2A gene and male infertility were evaluated for the first time. Materials and Methods: In this experimental study, 400 infertile men (oligospermia and azoospermia) and normal healthy fathers were evaluated (n=200). Single Strand Conformation Polymorphism (SSCP-PCR) and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods were used for screening any polymorphisms that are exist in exon 12 and exon 24. Results: Exon 24 PCR products were analyzed by RFLP but no polymorphism was found in this exon at the restriction site of EcoRV enzyme. Our monitoring along the whole nucleotides of exon 12 and exon 24 were continued using SSCP method, but we found no change along these exons. Conclusion: Generally, this study evaluated the association between polymorphisms in exon 12 and exon 24 of JHDM2A gene and male infertility which suggests that polymorphisms of these exons may not be associated with the risk of male infertility. PMID:27525322

  8. Noncoder: a web interface for exon array-based detection of long non-coding RNAs

    PubMed Central

    Gellert, Pascal; Ponomareva, Yuliya; Braun, Thomas; Uchida, Shizuka

    2013-01-01

    Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called ‘noncoder’. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de. PMID:23012263

  9. Evaluation of 2'-Deoxy-2'-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy.

    PubMed

    Jirka, Silvana M G; Tanganyika-de Winter, Christa L; Boertje-van der Meulen, Joke W; van Putten, Maaike; Hiller, Monika; Vermue, Rick; de Visser, Peter C; Aartsma-Rus, Annemieke

    2015-01-01

    Duchenne muscular dystrophy (DMD) is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs) that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2'-deoxy-2'-fluoro (2F) RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2'-substituted AONs (2'-F phosphorothioate (2FPS) and 2'-O-Me phosphorothioate (2OMePS)) on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON. PMID:26623937

  10. Exon circularization requires canonical splice signals.

    PubMed

    Starke, Stefan; Jost, Isabelle; Rossbach, Oliver; Schneider, Tim; Schreiner, Silke; Hung, Lee-Hsueh; Bindereif, Albrecht

    2015-01-01

    Circular RNAs (circRNAs), an abundant class of noncoding RNAs in higher eukaryotes, are generated from pre-mRNAs by circularization of adjacent exons. Using a set of 15 circRNAs, we demonstrated their cell-type-specific expression and circular versus linear processing in mammalian cells. Northern blot analysis combined with RNase H cleavage conclusively proved a circular configuration for two examples, LPAR1 and HIPK3. To address the circularization mechanism, we analyzed the sequence requirements using minigenes derived from natural circRNAs. Both canonical splice sites are required for circularization, although they vary in flexibility and potential use of cryptic sites. Surprisingly, we found that no specific circRNA exon sequence is necessary and that potential flanking intron structures can modulate circularization efficiency. In combination with splice inhibitor assays, our results argue that the canonical spliceosomal machinery functions in circRNA biogenesis, constituting an alternative splicing mode. PMID:25543144

  11. Foldons, Protein Structural Modules, and Exons

    NASA Astrophysics Data System (ADS)

    Panchenko, Anna R.; Luthey-Schulten, Zaida; Wolynes, Peter G.

    1996-03-01

    Foldons, which are kinetically competent, quasi-independently folding units of a protein, may be defined using energy landscape analysis. Foldons can be identified by maxima in a scan of the ratio of a contiguous segment's energetic stability gap to the energy variance of that segment's molten globule states, reflecting the requirement of minimal frustration. The predicted foldons are compared with the exons and structural modules for 16 of the 30 proteins studied. Statistical analysis indicates a strong correlation between the energetically determined foldons and Go's geometrically defined structural modules, but there are marked sequence-dependent effects. There is only a weak correlation of foldons to exons. For γ II-crystallin, myoglobin, barnase, α -lactalbumin, and cytochrome c the foldons and some noncontiguous clusters of foldons compare well with intermediates observed in experiment.

  12. Exonic splicing signals impose constraints upon the evolution of enzymatic activity.

    PubMed

    Falanga, Alessia; Stojanović, Ozren; Kiffer-Moreira, Tina; Pinto, Sofia; Millán, José Luis; Vlahoviček, Kristian; Baralle, Marco

    2014-05-01

    Exon splicing enhancers (ESEs) overlap with amino acid coding sequences implying a dual evolutionary selective pressure. In this study, we map ESEs in the placental alkaline phosphatase gene (ALPP), absent in the corresponding exon of the ancestral tissue-non-specific alkaline phosphatase gene (ALPL). The ESEs are associated with amino acid differences between the transcripts in an area otherwise conserved. We switched out the ALPP ESEs sequences with the sequence from the related ALPL, introducing the associated amino acid changes. The resulting enzymes, produced by cDNA expression, showed different kinetic characteristics than ALPL and ALPP. In the organism, this enzyme will never be subjected to selection because gene splicing analysis shows exon skipping due to loss of the ESE. Our data prove that ESEs restrict the evolution of enzymatic activity. Thus, suboptimal proteins may exist in scenarios when coding nucleotide changes and consequent amino acid variation cannot be reconciled with the splicing function. PMID:24692663

  13. Combinatorial control of a neuron-specific exon.

    PubMed Central

    Modafferi, E F; Black, D L

    1999-01-01

    The mouse c-src gene contains a short neuron-specific exon, N1. N1 exon splicing is partly controlled by an intronic splicing enhancer sequence that activates splicing of a heterologous reporter exon in both neural and nonneural cells. Here we attempt to dissect all of the regulatory elements controlling the N1 exon and examine how these multiple elements work in combination. We show that the 3' splice site sequence upstream of exon N1 represses the activation of splicing by the downstream intronic enhancer. This repression is stronger in nonneural cells and these two regulatory sequences combine to make a reporter exon highly cell-type specific. Substitution of the 3' splice site of this test exon with sites from other exons indicates that activation by the enhancer is very dependent on the nature of the upstream 3' splice site. In addition, we identify a previously uncharacterized purine-rich sequence within exon N1 that cooperates with the downstream intronic enhancer to increase exon inclusion. Finally, different regulatory elements were tested in multiple cell lines of both neuronal and nonneuronal origin. The individual splicing regulatory sequences from the src gene vary widely in their activity between different cell lines. These results demonstrate how a simple cassette exon is controlled by a variety of regulatory elements that only in combination will produce the correct tissue specificity of splicing. PMID:10334339

  14. Exon4 Amelogenin Transcripts in Enamel Biomineralization

    PubMed Central

    Stahl, J.; Nakano, Y.; Horst, J.; Zhu, L.; Le, M.; Zhang, Y.; Liu, H.; Li, W.

    2015-01-01

    Amelogenins are proteins formed by alternative splicing of the amelogenin gene, and are essential for tooth enamel formation. However, the unique functions of various alternatively spliced amelogenins in enamel formation are not well understood. In this study, we determined the spatiotemporal location of amelogenins derived from transcripts containing exon4 (AMG+4) in the enamel matrix, and the relative binding of recombinant AMG+4 to hydroxyapatite (HAP). Immunohistochemistry and mass spectrometry analyses showed that AMG+4 proteins were secreted into the enamel matrix at the early maturation stage. A stage-specific increase in the synthesis of AMG+4 was further supported by our observation that in mice overexpressing leucine-rich amelogenin peptide (TgLRAP), in which ameloblasts differentiate earlier, AMG+4 transcripts were also upregulated earlier. In vitro binding studies, supported by in silico modeling of protein binding to calcium and phosphate, showed that more recombinant AMG+4 bound to hydroxyapatite (HAP) as compared with recombinant AMG-4. The temporal and spatial localization of amelogenins containing exon4 peptide, and their functional differences in HAP binding, suggests that the unique properties of amelogenins containing exon4 cause a specific enhancement of biomineralization related to stabilization of early-formed HAP at the maturation stage. PMID:25792521

  15. Exon4 amelogenin transcripts in enamel biomineralization.

    PubMed

    Stahl, J; Nakano, Y; Horst, J; Zhu, L; Le, M; Zhang, Y; Liu, H; Li, W; Den Besten, P K

    2015-06-01

    Amelogenins are proteins formed by alternative splicing of the amelogenin gene, and are essential for tooth enamel formation. However, the unique functions of various alternatively spliced amelogenins in enamel formation are not well understood. In this study, we determined the spatiotemporal location of amelogenins derived from transcripts containing exon4 (AMG+4) in the enamel matrix, and the relative binding of recombinant AMG+4 to hydroxyapatite (HAP). Immunohistochemistry and mass spectrometry analyses showed that AMG+4 proteins were secreted into the enamel matrix at the early maturation stage. A stage-specific increase in the synthesis of AMG+4 was further supported by our observation that in mice overexpressing leucine-rich amelogenin peptide (TgLRAP), in which ameloblasts differentiate earlier, AMG+4 transcripts were also upregulated earlier. In vitro binding studies, supported by in silico modeling of protein binding to calcium and phosphate, showed that more recombinant AMG+4 bound to hydroxyapatite (HAP) as compared with recombinant AMG-4. The temporal and spatial localization of amelogenins containing exon4 peptide, and their functional differences in HAP binding, suggests that the unique properties of amelogenins containing exon4 cause a specific enhancement of biomineralization related to stabilization of early-formed HAP at the maturation stage. PMID:25792521

  16. Tissue-specific expression of the bovine aromatase-encoding gene uses multiple transcriptional start sites and alternative first exons.

    PubMed

    Fürbass, R; Kalbe, C; Vanselow, J

    1997-07-01

    Here we report on the genomic structure of the bovine aromatase cytochrome P450-encoding gene (Cyp19) and its tissue-specific transcript variants. The gene comprises at least 14 exons (1.1, 1.2a, 1.2b, 1.3,1.4, and 2-10) spanning more than 56 kilobases of genomic DNA. The coding area is confined to exons 2-10. Transcriptional start sites of Cyp19 were examined in granulosa cells, placenta, testis, adrenal gland, and brain, employing 5'-RACE (rapid amplification of complementary DNA ends) and primer extension. The analysis of 5'-RACE clones revealed six Cyp19 transcript variants that were different within their 5'-untranslated regions (5'-UTR). Yet, the coding region was identical in all clones. Although two of these 5'-UTR (the first 152 nucleotides of exon 2 and exon 1.4) are conserved among different species, four others (exons 1.1, 1.2a, 1.2b, and 1.3) did not show sequence homology to any other species. Transcription from exons 1.1 and 2 starts at several adjacent sites. In granulosa cells and placenta, but not in brain, a fraction of transcripts starting with exon 1.2a contains an additional untranslated exon, 1.2b, due to alternative splicing. Transcript variants comprising exon 1.1, 1.2a, 1.2b, or 1.3 were mainly found in the placenta, those with the 5'-UTR of exon 2 were predominant in granulosa cells, and transcripts with exon 1.4 prevailed in the brain. Estimates of Cyp19 transcript concentrations in six different tissues revealed high levels in granulosa cells and placenta, intermediate levels in testis and brain, and low levels in adrenal gland and liver. Our experiments demonstrate that six transcript variants of the bovine Cyp19 gene, including 9-11 exons, are expressed with tissue-specific preferences. These transcripts are presumably generated using five different promoter regions and tissue-specific alternative splicing. PMID:9202222

  17. Validation of Mismatch Negativity and P3a for Use in Multi-Site Studies of Schizophrenia: Characterization of Demographic, Clinical, Cognitive, and Functional Correlates in COGS-2

    PubMed Central

    Light, Gregory A.; Swerdlow, Neal R.; Thomas, Michael L.; Calkins, Monica E.; Green, Michael F.; Greenwood, Tiffany A.; Gur, Raquel E.; Gur, Ruben C.; Lazzeroni, Laura C.; Nuechterlein, Keith H.; Pela, Marlena; Radant, Allen D.; Seidman, Larry J.; Sharp, Richard F.; Siever, Larry J.; Silverman, Jeremy M.; Sprock, Joyce; Stone, William S.; Sugar, Catherine A.; Tsuang, Debby W.; Tsuang, Ming T.; Braff, David L.; Turetsky, Bruce I.

    2014-01-01

    Mismatch negativity (MMN) and P3a are auditory event-related potential (ERP) components that show robust deficits in schizophrenia (SZ) patients and exhibit qualities of endophenotypes, including substantial heritability, test-retest reliability, and trait-like stability. These measures also fulfill criteria for use as cognition and function-linked biomarkers in outcome studies, but have not yet been validated for use in large-scale multi-site clinical studies. This study tested the feasibility of adding MMN and P3a to the ongoing Consortium on the Genetics of Schizophrenia (COGS) study. The extent to which demographic, clinical, cognitive, and functional characteristics contribute to variability in MMN and P3a amplitudes was also examined. Participants (HCS n=824, SZ n=966) underwent testing at 5 geographically distributed COGS laboratories. Valid ERP data was obtained from 91% of HCS and 91% of SZ patients. Highly significant MMN (d=0.96) and P3a (d=0.93) amplitude reductions were observed in SZ patients, comparable in magnitude to those observed in single-lab studies with no appreciable differences across laboratories. Demographic characteristics accounted for 26% and 18% of the variance in MMN and P3a amplitudes, respectively. Significant relationships were observed among demographically-adjusted MMN and P3a measures and medication status as well as several clinical, cognitive, and functional characteristics of the SZ patients. This study demonstrates that MMN and P3a ERP biomarkers can be feasibly used in multi-site clinical studies. As with many clinical tests of brain function, demographic factors contribute to MMN and P3a amplitudes and should be carefully considered in future biomarker-informed clinical studies. PMID:25449710

  18. A general role for splicing enhancers in exon definition.

    PubMed Central

    Lam, Bianca J; Hertel, Klemens J

    2002-01-01

    Exonic splicing enhancers (ESEs) facilitate exon definition by assisting in the recruitment of splicing factors to the adjacent intron. Here we demonstrate that suboptimal 5' and 3' splice sites are activated independently by ESEs when they are located on different exons. However, when they are situated within a single exon, the same weak 5' and 3' splice sites are activated simultaneously by a single ESE. These findings demonstrate that a single ESE promotes the recognition of both exon/intron junctions within the same step during exon definition. Our results suggest that ESEs recruit a multicomponent complex that minimally contains components of the splicing machinery required for 5' and 3' splice site selection. PMID:12403462

  19. Exonic Variants Associated with Development of Aspirin Exacerbated Respiratory Diseases

    PubMed Central

    Chang, HunSoo; Park, Jong Sook; Bae, Da-Jeong; Song, Hyun-Ji; Choi, Inseon S.; Kim, Mi-Kyeong; Park, Hea-Sim; Kim, Lyoung Hyo; Namgoong, Suhg; Kim, Ji On; Shin, Hyoung Doo; Park, Choon-Sik

    2014-01-01

    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. PMID:25372592

  20. Dual Masking of Specific Negative Splicing Regulatory Elements Resulted in Maximal Exon 7 Inclusion of SMN2 Gene

    PubMed Central

    Pao, Peng Wen; Wee, Keng Boon; Yee, Woon Chee; DwiPramono, Zacharias Aloysius

    2014-01-01

    Spinal muscular atrophy (SMA) is a fatal autosomal recessive disease caused by survival motor neuron (SMN) protein insufficiency due to SMN1 mutations. Boosting SMN2 expression is a potential therapy for SMA. SMN2 has identical coding sequence as SMN1 except for a silent C-to-T transition at the 6th nucleotide of exon 7, converting a splicing enhancer to a silencer motif. Consequently, most SMN2 transcripts lack exon 7. More than ten putative splicing regulatory elements (SREs) were reported to regulate exon 7 splicing. To investigate the relative strength of each negative SRE in inhibiting exon 7 inclusion, antisense oligonucleotides (AONs) were used to mask each element, and the fold increase of full-length SMN transcripts containing exon 7 were compared. The most potent negative SREs are at intron 7 (in descending order): ISS-N1, 3′ splice site of exon 8 (ex8 3′ss) and ISS+100. Dual-targeting AONs were subsequently used to mask two nonadjacent SREs simultaneously. Notably, masking of both ISS-N1 and ex8 3′ss induced the highest fold increase of full-length SMN transcripts and proteins. Therefore, efforts should be directed towards the two elements simultaneously for the development of optimal AONs for SMA therapy. PMID:24317636

  1. Effects of Blocking D2/D3 Receptors on Mismatch Negativity and P3a Amplitude of Initially Antipsychotic Naïve, First Episode Schizophrenia Patients

    PubMed Central

    Glenthøj, Birte Yding; Oranje, Bob

    2016-01-01

    Background: Reduced mismatch negativity and P3a amplitude have been suggested to be among the core deficits in schizophrenia since the late 1970s. Blockade of dopamine D2 receptors play an important role in the treatment of schizophrenia. In addition, there is some evidence indicating that deficits in mismatch negativity and P3a amplitude are related to increased dopaminergic activity. This is the first study investigating the effect of amisulpride, a potent D2-antagonist, on mismatch negativity and P3a amplitude in a large group of antipsychotic-naïve, first-episode schizophrenia patients. Methods: Fifty-one antipsychotic-naïve, first-episode schizophrenia patients were tested in a mismatch negativity paradigm at baseline and after 6 weeks of treatment with amisulpride. We further examined 48 age- and gender-matched controls in this paradigm. Results: At baseline, the patients showed significantly reduced P3a amplitude compared with healthy controls, but no differences in mismatch negativity. Although the treatment with amisulpride significantly improved the patients’ psychopathological (PANSS) and functional (GAF) scores, it did not influence their mismatch negativity amplitude, while also their reduced P3a amplitude persisted. Conclusion: Our findings show that antipsychotic naïve, first-episode patients with schizophrenia have normal mismatch negativity yet reduced P3a amplitude compared with healthy controls. In spite of the fact that the 6-week amisulpride treatment improved the patients both clinically and functionally, it had no effect on either mismatch negativity or P3a amplitude. This suggests that even though there is a dopaminergic involvement in global functioning and symptomatology in schizophrenia, there is no such involvement in these particular measures of early information processing. PMID:26453696

  2. A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

    PubMed Central

    Humphrey, M B; Bryan, J; Cooper, T A; Berget, S M

    1995-01-01

    Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon. PMID:7623794

  3. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy.

    PubMed

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  4. Abnormal splicing switch of DMD's penultimate exon compromises muscle fibre maintenance in myotonic dystrophy

    PubMed Central

    Rau, Frédérique; Lainé, Jeanne; Ramanoudjame, Laetitita; Ferry, Arnaud; Arandel, Ludovic; Delalande, Olivier; Jollet, Arnaud; Dingli, Florent; Lee, Kuang-Yung; Peccate, Cécile; Lorain, Stéphanie; Kabashi, Edor; Athanasopoulos, Takis; Koo, Taeyoung; Loew, Damarys; Swanson, Maurice S.; Le Rumeur, Elisabeth; Dickson, George; Allamand, Valérie; Marie, Joëlle; Furling, Denis

    2015-01-01

    Myotonic Dystrophy type 1 (DM1) is a dominant neuromuscular disease caused by nuclear-retained RNAs containing expanded CUG repeats. These toxic RNAs alter the activities of RNA splicing factors resulting in alternative splicing misregulation and muscular dysfunction. Here we show that the abnormal splicing of DMD exon 78 found in dystrophic muscles of DM1 patients is due to the functional loss of MBNL1 and leads to the re-expression of an embryonic dystrophin in place of the adult isoform. Forced expression of embryonic dystrophin in zebrafish using an exon-skipping approach severely impairs the mobility and muscle architecture. Moreover, reproducing Dmd exon 78 missplicing switch in mice induces muscle fibre remodelling and ultrastructural abnormalities including ringed fibres, sarcoplasmic masses or Z-band disorganization, which are characteristic features of dystrophic DM1 skeletal muscles. Thus, we propose that splicing misregulation of DMD exon 78 compromises muscle fibre maintenance and contributes to the progressive dystrophic process in DM1. PMID:26018658

  5. Smooth muscle-specific switching of alpha-tropomyosin mutually exclusive exon selection by specific inhibition of the strong default exon.

    PubMed

    Gooding, C; Roberts, G C; Moreau, G; Nadal-Ginard, B; Smith, C W

    1994-08-15

    Exons 2 and 3 of alpha-tropomyosin are spliced in a strict mutually exclusive manner. Exon 3 is a default choice, being selected in almost all cell types where the gene is expressed. The default selection arises from a competition between the two exons, in which the stronger branch point/pyrimidine tract elements of exon 3 win. Exon 2 is selected predominantly or exclusively only in smooth muscle cells. We show here that the basis for the smooth muscle-specific switching of exon selection is inhibition of exon 3. Exon 3 is still skipped with smooth muscle specificity, even in the absence of exon 2. We have defined two conserved sequence elements, one in each of the introns flanking exon 3, that are essential for this regulation. Mutation of either element severely impairs regulated suppression of exon 3. No other exon or intron sequences appear to be necessary for regulation. We have also demonstrated skipping of exon 3 that is dependent upon both regulatory elements in an in vitro splicing assay. We further show that both splice sites of exon 3 must be inhibited in a concerted fashion to switch to selection of exon 2. This may relate to the requirement for negative elements on both sides of the exon. PMID:8070413

  6. Auditory P3a and P3b neural generators in schizophrenia: An adaptive sLORETA P300 localization approach.

    PubMed

    Bachiller, Alejandro; Romero, Sergio; Molina, Vicente; Alonso, Joan F; Mañanas, Miguel A; Poza, Jesús; Hornero, Roberto

    2015-12-01

    The present study investigates the neural substrates underlying cognitive processing in schizophrenia (Sz) patients. To this end, an auditory 3-stimulus oddball paradigm was used to identify P3a and P3b components, elicited by rare-distractor and rare-target tones, respectively. Event-related potentials (ERP) were recorded from 31 Sz patients and 38 healthy controls. The P3a and P3b brain-source generators were identified by time-averaging of low-resolution brain electromagnetic tomography (LORETA) current density images. In contrast with the commonly used fixed window of interest (WOI), we proposed to apply an adaptive WOI, which takes into account subjects' P300 latency variability. Our results showed different P3a and P3b source activation patterns in both groups. P3b sources included frontal, parietal and limbic lobes, whereas P3a response generators were localized over bilateral frontal and superior temporal regions. These areas have been related to the discrimination of auditory stimulus and to the inhibition (P3a) or the initiation (P3b) of motor response in a cognitive task. In addition, differences in source localization between Sz and control groups were observed. Sz patients showed lower P3b source activity in bilateral frontal structures and the cingulate. P3a generators were less widespread for Sz patients than for controls in right superior, medial and middle frontal gyrus. Our findings suggest that target and distractor processing involves distinct attentional subsystems, both being altered in Sz. Hence, the study of neuroelectric brain information can provide further insights to understand cognitive processes and underlying mechanisms in Sz. PMID:26481687

  7. Preattentive sensory processing as indexed by the MMN and P3a brain responses is associated with cognitive and psychosocial functioning in healthy adults.

    PubMed

    Light, Gregory A; Swerdlow, Neal R; Braff, David L

    2007-10-01

    Understanding the basic neural processes that underlie complex higher order cognitive operations and psychosocial functioning is a fundamental goal of cognitive neuroscience. Event-related potentials allow investigators to probe the earliest stages of information processing. Mismatch negativity (MMN) and P3a are auditory event-related potential components that reflect automatic sensory discrimination. The aim of the present study was to determine if MMN and P3a are associated with higher order cognitive operations and psychosocial functioning in clinically normal healthy subjects. Twenty adults were assessed using standardized clinical, cognitive, and psychosocial functional instruments. All individuals were within the normal range on cognitive tests and functional ratings. Participants were also tested on a duration-deviant MMN/P3a paradigm (50-msec standard tones, p = .90; 100-msec deviant tones, p = .10; stimulus onset asynchrony [SOA] = 505 msec). Across fronto-central electrode regions, significant correlations were observed between psychosocial functioning and MMN (r = -.62, p < .01) and P3a (r = .63, p < .01) amplitudes. P3a amplitude was also highly associated with immediate and delayed recall of verbal information with robust correlations widely distributed across fronto-central recording areas (e.g., r = .72, p < .001). The latency of the P3a response was significantly associated with both working memory performance (r = -.53, p < .05) and functional ratings (r = -.48, p < .05). Neurophysiological measures of relatively automatic auditory sensory information processing are associated with higher order cognitive abilities and psychosocial functioning in normal subjects. Efficiency at elementary levels of information processing may underlie the successful encoding, retrieval, and discrimination of task-relevant information, which, in turn, facilitates the iterative and responsive processing necessary for adaptive cognitive and social functioning. PMID

  8. A single-nucleotide exon found in Arabidopsis

    PubMed Central

    Guo, Lei; Liu, Chun-Ming

    2015-01-01

    The presence of introns in gene-coding regions is one of the most mysterious evolutionary inventions in eukaryotic organisms. It has been proposed that, although sequences involved in intron recognition and splicing are mainly located in introns, exonic sequences also contribute to intron splicing. The smallest constitutively spliced exon known so far has 6 nucleotides, and the smallest alternatively spliced exon has 3 nucleotides. Here we report that the Anaphase Promoting Complex subunit 11 (APC11) gene in Arabidopsis thaliana carries a constitutive single-nucleotide exon. In vivo transcription and translation assays performed using APC11-Green Fluorescence Protein (GFP) fusion constructs revealed that intron splicing surrounding the single-nucleotide exon is effective in both Arabidopsis and rice. This discovery warrants attention to genome annotations in the future. PMID:26657562

  9. A single-nucleotide exon found in Arabidopsis.

    PubMed

    Guo, Lei; Liu, Chun-Ming

    2015-01-01

    The presence of introns in gene-coding regions is one of the most mysterious evolutionary inventions in eukaryotic organisms. It has been proposed that, although sequences involved in intron recognition and splicing are mainly located in introns, exonic sequences also contribute to intron splicing. The smallest constitutively spliced exon known so far has 6 nucleotides, and the smallest alternatively spliced exon has 3 nucleotides. Here we report that the Anaphase Promoting Complex subunit 11 (APC11) gene in Arabidopsis thaliana carries a constitutive single-nucleotide exon. In vivo transcription and translation assays performed using APC11-Green Fluorescence Protein (GFP) fusion constructs revealed that intron splicing surrounding the single-nucleotide exon is effective in both Arabidopsis and rice. This discovery warrants attention to genome annotations in the future. PMID:26657562

  10. A premature termination codon within an alternative exon affecting only the metabolism of transcripts that retain this exon.

    PubMed

    Maillet, P; Dalla Venezia, N; Lorenzo, F; Morinière, M; Bozon, M; Noël, B; Delaunay, J; Baklouti, F

    1999-01-01

    Protein 4.1 pre-mRNA splicing is regulated in tissue- and development-specific manners. Exon 16, which encodes the N-terminal region of the spectrin/actin-binding domain, is one of the alternatively spliced sequence motifs. It is present in late differentiated erythroid cells but absent from early erythroblasts and from lymphoid cells. We describe a single nucleotide deletion of the erythroid protein 4.1 gene associated with hereditary elliptocytosis. The deletion located in exon 16 leads to a frameshift and a premature termination codon within the same exon. In an effort to examine the premature stop codon effect in relationship with exon 16 alternative splicing, we analyzed erythroid and lymphoid protein 4.1 mRNAs using the mutation and a linked downstream polymorphism as markers. We found that the premature stop codon does not affect the tissue-specific alternative splicing among the two cell types analyzed and that the resulting alteration of mRNA metabolism correlates with the retention of exon 16 in reticulocytes. Conversely, skipping of exon 16 in lymphoid cells converts the mutant mRNA to a normal lymphoid-specific mRNA isoform, hence bypassing the nonsense codon. Consistent with data obtained on constitutive nonsense exons, our observations argue in favor of a stop codon recognition mechanism that occurs after the regulated splicing status of the nonsense exon has been achieved. PMID:10425037

  11. Alcohol abuse and HIV infection have additive effects on frontal cortex function as measured by auditory evoked potential P3A latency.

    PubMed

    Fein, G; Biggins, C A; MacKay, S

    1995-02-01

    Both alcohol and human immunodeficiency virus (HIV) infection have been shown to produce central nervous system (CNS) morbidity in frontal brain regions. The degree to which the CNS morbidity in HIV infection, as it affects frontal cortex function, may be preferentially increased by alcohol abuse was examined using the auditory P3A evoked potential. The P3A indexes an orienting response, maximal over frontal cortex that occurs when novel nontarget stimuli are presented in the midst of a target detection paradigm. Four groups of subjects were compared: HIV+ alcohol abusers, HIV+ light/nondrinkers, HIV- alcohol abusers, and HIV- light/nondrinkers. The alcohol abuser and light/nondrinker HIV+ groups were matched on percent CD4 lymphocytes, insuring that the results reflected specific CNS effects and were not a result of differences between the groups in the degree of systemic immune suppression. Alcohol abuse and HIV infection had at least additive effects on P3A latency, consistent with alcohol abuse worsening the effect of HIV disease on frontal cortex function. Post-hoc analyses suggested that concomitant alcohol abuse results in the effects of HIV infection on P3A latency becoming manifest earlier in the HIV disease process. PMID:7727627

  12. Regulation of alternative splicing of tau exon 10.

    PubMed

    Qian, Wei; Liu, Fei

    2014-04-01

    The neuronal microtubule-associated protein tau is abnormally hyperphosphorylated and aggregated into neurofibrillary tangles in the brains of individuals with Alzheimer's disease and related neurodegenerative disorders. The adult human brain expresses six isoforms of tau generated by alternative splicing of exons 2, 3, and 10 of its pre-mRNA. Exon 10 encodes the second microtubule-binding repeat of tau. Its alternative splicing produces tau isoforms with either three or four microtubule-binding repeats, termed 3R-tau and 4Rtau. In the normal adult human brain, the level of 3R-tau is approximately equal to that of 4R-tau. Several silent and intronic mutations of the tau gene associated with FTDP-17T (frontotemporal dementia with Parkinsonism linked to chromosome 17 and specifically characterized by tau pathology) only disrupt exon 10 splicing, but do not influence the primary sequence of the tau protein. Thus, abnormal exon 10 splicing is sufficient to cause neurodegeneration and dementia. Here, we review the regulation of tau exon 10 splicing by cis-elements and trans-factors and summarize all the mutations associated with FTDP-17T and related tauopathies. The findings suggest that correction of exon 10 splicing may be a potential target for tau exon 10 splicing-related tauopathies. PMID:24627328

  13. Switching Attention Within Working Memory is Reflected in the P3a Component of the Human Event-Related Brain Potential

    PubMed Central

    Berti, Stefan

    2016-01-01

    The flexible access to information in working memory is crucial for adaptive behavior. It is assumed that this is realized by switching the focus of attention within working memory. Switching of attention is mirrored in the P3a component of the human event-related brain potential (ERP) and it has been argued that the processes reflected by the P3a are also relevant for selecting information within working memory. The aim of the present study was to further evaluate whether the P3a mirrors genuine switching of attention within working memory by applying an object switching task: Participants updated a memory list of four digits either by replacing one item with another digit or by processing the stored digit. ERPs were computed separately for two types of trials: (1) trials in which an object was repeated and (2) trials in which a switch to a new object was required in order to perform the task. Object-switch trials showed increased response times compared with repetition trials in both task conditions. In addition, switching costs were increased in the processing compared with the replacement condition. Pronounced P3a’s were obtained in switching trials but there were no difference between the two updating tasks (replacement or processing). These results were qualified by the finding that the magnitude of the visual location shift also affects the ERPs in the P3a time window. Taken together, the present pattern of results suggest that the P3a reflects an initial process of selecting information in working memory but not the memory updating itself. PMID:26779009

  14. A Duplication in the Canine β-Galactosidase Gene GLB1 Causes Exon Skipping and GM1-Gangliosidosis in Alaskan Huskies

    PubMed Central

    Kreutzer, Robert; Leeb, Tosso; Müller, Gundi; Moritz, Andreas; Baumgärtner, Wolfgang

    2005-01-01

    GM1-gangliosidosis is a lysosomal storage disease that is inherited as an autosomal recessive disorder, predominantly caused by structural defects in the β-galactosidase gene (GLB1). The molecular cause of GM1-gangliosidosis in Alaskan huskies was investigated and a novel 19-bp duplication in exon 15 of the GLB1 gene was identified. The duplication comprised positions +1688–+1706 of the GLB1 cDNA. It partially disrupted a potential exon splicing enhancer (ESE), leading to exon skipping in a fraction of the transcripts. Thus, the mutation caused the expression of two different mRNAs from the mutant allele. One transcript contained the complete exon 15 with the 19-bp duplication, while the other transcript lacked exon 15. In the transcript containing exon 15 with the 19-bp duplication a premature termination codon (PTC) appeared, but due to its localization in the last exon of canine GLB1, nonsense-mediated RNA decay (NMD) did not occur. As a consequence of these molecular events two different truncated GLB1 proteins are predicted to be expressed from the mutant GLB1 allele. In heterozygous carrier animals the wild-type allele produces sufficient amounts of the active enzyme to prevent clinical signs of disease. In affected homozygous dogs no functional GLB1 is synthesized and GM1-gangliosidosis occurs. PMID:15944348

  15. Exonic Mutations in the SLC12A3 Gene Cause Exon Skipping and Premature Termination in Gitelman Syndrome

    PubMed Central

    Takeuchi, Yoichi; Mishima, Eikan; Shima, Hisato; Akiyama, Yasutoshi; Suzuki, Chitose; Suzuki, Takehiro; Kobayashi, Takayasu; Suzuki, Yoichi; Nakayama, Tomohiro; Takeshima, Yasuhiro; Vazquez, Norma; Ito, Sadayoshi; Gamba, Gerardo

    2015-01-01

    A variety of genetic backgrounds cause the loss of function of thiazide-sensitive sodium chloride cotransporter, encoded by SLC12A3, responsible for the phenotypes in Gitelman syndrome. Recently, the phenomenon of exon skipping, in which exonic mutations result in abnormal splicing, has been associated with various diseases. Specifically, mutations in exonic splicing enhancer (ESE) sequences can promote exon skipping. Here, we used a bioinformatics program to analyze 88 missense mutations in the SLC12A3 gene and identify candidate mutations that may induce exon skipping. The three candidate mutations that reduced ESE scores the most were further investigated by minigene assay, and two (p.A356V and p.M672I) caused abnormal splicing in vitro. Furthermore, we identified the p.M672I (c.2016G>A) mutation in a patient with Gitelman syndrome and found that this single nucleotide mutation causes exclusion of exon 16 in the SLC12A3 mRNA transcript. Functional analyses revealed that the protein encoded by the aberrant SLC12A3 transcript does not transport sodium. These results suggest that aberrant exon skipping is one previously unrecognized mechanism by which missense mutations in SLC12A3 can lead to Gitelman syndrome. PMID:25060058

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

    PubMed Central

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M.; Krishnaswarmy, Sudarsan; Wong, Brenda L.; Fletcher, Sue; Wilton, Steve D.

    2016-01-01

    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. PMID:26745801

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

    PubMed

    Toh, Zhi Yon Charles; Thandar Aung-Htut, May; Pinniger, Gavin; Adams, Abbie M; Krishnaswarmy, Sudarsan; Wong, Brenda L; Fletcher, Sue; Wilton, Steve D

    2016-01-01

    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. PMID:26745801

  18. Human glucose phosphate isomerase: Exon mapping and gene structure

    SciTech Connect

    Xu, Weiming; Lee, Pauline; Beutler, E.

    1995-10-10

    The structure of the gene for human glucose phosphate isomerase (GPI) has been determined. Three GPI clones were isolated from a human genomic library by using a full-length GPI cDNA probe and were characterized. Oligonucleotides based on the known cDNA sequence were used as primers in amplification and sequence analyses. This led to the identification of the exon-intron junctions. By this approach, 18 exons and 17 introns have been identified. The exons range in size from 44 to 431 nucleotides. The intronic sequences surrounding the exons provide useful information for the identification of mutations that give rise to human GPI deficiency associated with chronic hemolytic anemia. 13 refs., 4 figs., 1 tab.

  19. Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties

    PubMed Central

    2012-01-01

    Background For decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described. Results From the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties. Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation. Conclusions An Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse

  20. Identification of human chromosome 9 specific genes using exon amplification.

    PubMed

    Church, D M; Banks, L T; Rogers, A C; Graw, S L; Housman, D E; Gusella, J F; Buckler, A J

    1993-11-01

    We have recently developed a method, exon amplification, that is designed for isolation of exon sequences from genomic DNA. To assess the efficacy of this method we have analyzed cosmid genomic clones derived from human chromosome 9, and have cloned several products from this analysis. Approximately 63% of cosmids produced at least one product derived from functioning splice sites within the target genomic fragment, and in many cases multiple products were isolated. In addition, an easily identifiable class of false positives was produced from 56% of cosmids analyzed; these are readily eliminated from subsequent study. Sequence analysis and database searches revealed that the majority (87%) of the putative exon clones were unique, the remainder being derived from repetitive sequences. Analysis of sequence conservation by Southern blotting in addition to cDNA screening experiments suggested that most, if not all, of these unique sequences represent true exons. The results of these studies indicate that exon amplification is a rapid and reliable approach for isolation of exon sequences from mammalian genomic DNA. PMID:7506603

  1. [Exon-skipping therapy for Duchenne muscular dystrophy].

    PubMed

    Takeda, Shin'ichi

    2011-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin expression. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin at the sarcolamma of dystrophic dogs, and improved phenotypes of affected dogs without serious side effects (Ann Neurol. 65: 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique. We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx 52 mice and found the amelioration of the phenotypes (Mol Ther, 2010). Clinical trials of exon 51 skipping for DMD patients is now going in our country and application of antisense strategy to other hereditary neuromuscular diseases is largely expected. PMID:22277414

  2. Intronic motif pairs cooperate across exons to promote pre-mRNA splicing

    PubMed Central

    2010-01-01

    Background A very early step in splice site recognition is exon definition, a process that is as yet poorly understood. Communication between the two ends of an exon is thought to be required for this step. We report genome-wide evidence for exons being defined through the combinatorial activity of motifs located in flanking intronic regions. Results Strongly co-occurring motifs were found to specifically reside in four intronic regions surrounding a large number of human exons. These paired motifs occur around constitutive and alternative exons but not pseudo exons. Most co-occurring motifs are limited to intronic regions within 100 nucleotides of the exon. They are preferentially associated with weaker exons. Their pairing is conserved in evolution and they exhibit a lower frequency of single nucleotide polymorphism when paired. Paired motifs display specificity with respect to distance from the exon borders and in constitutive versus alternative splicing. Many resemble binding sites for heterogeneous nuclear ribonucleoproteins. Specific pairs are associated with tissue-specific genes, the higher expression of which coincides with that of the pertinent RNA binding proteins. Tested pairs acted synergistically to enhance exon inclusion, and this enhancement was found to be exon-specific. Conclusions The exon-flanking sequence pairs identified here by genomic analysis promote exon inclusion and may play a role in the exon definition step in pre-mRNA splicing. We propose a model in which multiple concerted interactions are required between exonic sequences and flanking intronic sequences to effect exon definition. PMID:20704715

  3. Isolated exon 8 deletion in type 1 spinal muscular atrophy with bilateral optic atrophy: unusual genetic mutation leading to unusual manifestation?

    PubMed

    Maiti, D; Bhattacharya, M; Yadav, S

    2012-01-01

    Proximal spinal muscular atrophy (SMA) or type 1 SMA is a fatal autosomal recessive disorder usually caused by homozygous deletion of exons 7 and 8 in the survivor motor neuron (SMN) gene. Additional deletion of the neuronal apotosis inhibitory protein (NAIP) gene exacerbates the clinical severity. Isolated exon 8 deletion has been reported in a single case series of SMA types 2 and 3 and never with SMA type 1. While extraocular muscles are typically spared, there are a few case reports documenting associated external ophthalmoplegia. Optic atrophy is a hitherto unreported association of SMA. We report a 10-month-old male infant with SMA type 1 with optic atrophy due to isolated deletion of exon 8 of the SMN gene with intact exon 7 and NAIP gene. PMID:23298926

  4. AltAnalyze and DomainGraph: analyzing and visualizing exon expression data.

    PubMed

    Emig, Dorothea; Salomonis, Nathan; Baumbach, Jan; Lengauer, Thomas; Conklin, Bruce R; Albrecht, Mario

    2010-07-01

    Alternative splicing is an important mechanism for increasing protein diversity. However, its functional effects are largely unknown. Here, we present our new software workflow composed of the open-source application AltAnalyze and the Cytoscape plugin DomainGraph. Both programs provide an intuitive and comprehensive end-to-end solution for the analysis and visualization of alternative splicing data from Affymetrix Exon and Gene Arrays at the level of proteins, domains, microRNA binding sites, molecular interactions and pathways. Our software tools include easy-to-use graphical user interfaces, rigorous statistical methods (FIRMA, MiDAS and DABG filtering) and do not require prior knowledge of exon array analysis or programming. They provide new methods for automatic interpretation and visualization of the effects of alternative exon inclusion on protein domain composition and microRNA binding sites. These data can be visualized together with affected pathways and gene or protein interaction networks, allowing a straightforward identification of potential biological effects due to alternative splicing at different levels of granularity. Our programs are available at http://www.altanalyze.org and http://www.domaingraph.de. These websites also include extensive documentation, tutorials and sample data. PMID:20513647

  5. Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy

    PubMed Central

    Gramlich, Michael; Pane, Luna Simona; Zhou, Qifeng; Chen, Zhifen; Murgia, Marta; Schötterl, Sonja; Goedel, Alexander; Metzger, Katja; Brade, Thomas; Parrotta, Elvira; Schaller, Martin; Gerull, Brenda; Thierfelder, Ludwig; Aartsma-Rus, Annemieke; Labeit, Siegfried; Atherton, John J; McGaughran, Julie; Harvey, Richard P; Sinnecker, Daniel; Mann, Matthias; Laugwitz, Karl-Ludwig; Gawaz, Meinrad Paul; Moretti, Alessandra

    2015-01-01

    Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM. PMID:25759365

  6. Exon junction complex subunits are required to splice Drosophila MAP kinase, a large heterochromatic gene

    PubMed Central

    Roignant, Jean-Yves; Treisman, Jessica E.

    2010-01-01

    Summary The exon junction complex (EJC) is assembled on spliced mRNAs upstream of exon-exon junctions, and can regulate their subsequent translation, localization, or degradation. We isolated mutations in Drosophila mago nashi (mago), which encodes a core EJC subunit, based on their unexpectedly specific effects on photoreceptor differentiation. Loss of Mago prevents Epidermal growth factor receptor signaling, due to a large reduction in MAPK mRNA levels. MAPK expression also requires the EJC subunits Y14 and eIF4AIII, and EJC-associated splicing factors. Mago depletion does not affect the transcription or stability of MAPK mRNA, but alters its splicing pattern. MAPK expression from an exogenous promoter requires Mago only when the template includes introns. MAPK is the primary functional target of mago in eye development; in cultured cells, Mago knockdown disproportionately affects other large genes located in heterochromatin. These data support a nuclear role for EJC components in splicing a specific subset of introns. PMID:20946982

  7. Macaca specific exon creation event generates a novel ZKSCAN5 transcript.

    PubMed

    Kim, Young-Hyun; Choe, Se-Hee; Song, Bong-Seok; Park, Sang-Je; Kim, Myung-Jin; Park, Young-Ho; Yoon, Seung-Bin; Lee, Youngjeon; Jin, Yeung Bae; Sim, Bo-Woong; Kim, Ji-Su; Jeong, Kang-Jin; Kim, Sun-Uk; Lee, Sang-Rae; Park, Young-Il; Huh, Jae-Won; Chang, Kyu-Tae

    2016-02-15

    ZKSCAN5 (also known as ZFP95) is a zinc-finger protein belonging to the Krűppel family. ZKSCAN5 contains a SCAN box and a KRAB A domain and is proposed to play a distinct role during spermatogenesis. In humans, alternatively spliced ZKSCAN5 transcripts with different 5'-untranslated regions (UTRs) have been identified. However, investigation of our Macaca UniGene Database revealed novel alternative ZKSCAN5 transcripts that arose due to an exon creation event. Therefore, in this study, we identified the full-length sequences of ZKSCAN5 and its alternative transcripts in Macaca spp. Additionally, we investigated different nonhuman primate sequences to elucidate the molecular mechanism underlying the exon creation event. We analyzed the evolutionary features of the ZKSCAN5 transcripts by reverse transcription polymerase chain reaction (RT-PCR) and genomic PCR, and by sequencing various nonhuman primate DNA and RNA samples. The exon-created transcript was only detected in the Macaca lineage (crab-eating monkey and rhesus monkey). Full-length sequence analysis by rapid amplification of cDNA ends (RACE) identified ten full-length transcripts and four functional isoforms of ZKSCAN5. Protein sequence analyses revealed the presence of two groups of isoforms that arose because of differences in start-codon usage. Together, our results demonstrate that there has been specific selection for a discrete set of ZKSCAN5 variants in the Macaca lineage. Furthermore, study of this locus (and perhaps others) in Macaca spp. might facilitate our understanding of the evolutionary pressures that have shaped the mechanism of exon creation in primates. PMID:26657034

  8. Recombinant Exon-Encoded Resilins for Elastomeric Biomaterials

    PubMed Central

    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

  9. Recombinant exon-encoded resilins for elastomeric biomaterials.

    PubMed

    Qin, Guokui; Rivkin, Amit; Lapidot, Shaul; Hu, Xiao; Preis, Itan; Arinus, Shira B; Dgany, Or; Shoseyov, Oded; Kaplan, David L

    2011-12-01

    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-mediated di-tyrosine 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 di-tyrosine 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

  10. Ameliorating pathogenesis by removing an exon containing a missense mutation: a potential exon-skipping therapy for laminopathies.

    PubMed

    Scharner, J; Figeac, N; Ellis, J A; Zammit, P S

    2015-06-01

    Exon skipping, as a therapy to restore a reading frame or switch protein isoforms, is under clinical trial. We hypothesised that removing an in-frame exon containing a mutation could also improve pathogenic phenotypes. Our model is laminopathies: incurable tissue-specific degenerative diseases associated with LMNA mutations. LMNA encodes A-type lamins, that together with B-type lamins, form the nuclear lamina. Lamins contain an alpha-helical central rod domain composed of multiple heptad repeats. Eliminating LMNA exon 3 or 5 removes six heptad repeats, so shortens, but should not otherwise significantly alter, the alpha-helix. Human Lamin A or Lamin C with a deletion corresponding to amino acids encoded by exon 5 (Lamin A/C-Δ5) localised normally in murine lmna-null cells, rescuing both nuclear shape and endogenous Lamin B1/emerin distribution. However, Lamin A carrying pathogenic mutations in exon 3 or 5, or Lamin A/C-Δ3, did not. Furthermore, Lamin A/C-Δ5 was not deleterious to wild-type cells, unlike the other Lamin A mutants including Lamin A/C-Δ3. Thus Lamin A/C-Δ5 function as effectively as wild-type Lamin A/C and better than mutant versions. Antisense oligonucleotides skipped LMNA exon 5 in human cells, demonstrating the possibility of treating certain laminopathies with this approach. This proof-of-concept is the first to report the therapeutic potential of exon skipping for diseases arising from missense mutations. PMID:25832542

  11. Exon capture phylogenomics: efficacy across scales of divergence.

    PubMed

    Bragg, Jason G; Potter, Sally; Bi, Ke; Moritz, Craig

    2016-09-01

    The evolutionary histories of species are not measured directly, but estimated using genealogies inferred for particular loci. Individual loci can have discordant histories, but in general we expect to infer evolutionary histories more accurately as more of the genome is sampled. High Throughput Sequencing (HTS) is now providing opportunities to incorporate thousands of loci in 'phylogenomic' studies. Here, we used target enrichment to sequence c.3000 protein-coding exons in a group of Australian skink lizards (crown group age c.80 Ma). This method uses synthetic probes to 'capture' target exons that were identified in the transcriptomes of selected probe design (PD) samples. The target exons are then enriched in sample DNA libraries prior to performing HTS. Our main goal was to study the efficacy of enrichment of targeted loci at different levels of phylogenetic divergence from the PD species. In taxa sharing a common ancestor with PD samples up to c.20 Ma, we detected little reduction in efficacy, measured here as sequencing depth of coverage. However, at around 80 Myr divergence from the PD species, we observed an approximately two-fold reduction in efficacy. A secondary goal was to develop a workflow for analysing exon capture studies of phylogenetically diverse samples, while minimizing potential bias. Our approach assembles each exon in each sample separately, by first recruiting short sequencing reads having homology to the corresponding protein sequence. In sum, custom exon capture provides a complement to existing, more generic target capture methods and is a practical and robust option across low-moderate levels of phylogenetic divergence. PMID:26215687

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

    PubMed

    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 promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA). PMID:21686247

  13. The Levels of Tau Isoforms Containing Exon-2 and Exon-10 Segments Increased in the Cerebrospinal Fluids of the Patients with Sporadic Creutzfeldt-Jakob Disease.

    PubMed

    Chen, Cao; Zhou, Wei; Lv, Yan; Shi, Qi; Wang, Jing; Xiao, Kang; Chen, Li-Na; Zhang, Bao-Yun; Dong, Xiao-Ping

    2016-08-01

    The alteration of protein tau in the cerebrospinal fluid (CSF) of Creutzfeldt-Jakob disease (CJD) has been widely evaluated, possessing a significant diagnostic value for CJD. With the biotin-labeled tau-exon-specific mAbs, direct ELISA methods were established and the levels of tau isoforms containing exon-2 and exon-10 segments in CSF of the patients with various human prion diseases and in brain tissues of scrapie-infected animals were evaluated. The results showed that the levels of tau, especially containing four repeats in microtubule binding domain, were increased in the CSF samples of the patients with sporadic CJD (sCJD). Using the unlabeled (cold) mixed exon-specific mAbs, a competitive tau ELISA was conducted based on a commercial tau kit. It revealed that the majority of the increased tau in the CSF of sCJD cases was derived from the tau isoforms with exon-2 and exon-10 segments. Increases of CSF tau isoforms with exon-2 and exon-10 segments were also observed in the patients of E200K and T188K genetic CJD (gCJD), but not in the cases of fatal familiar insomnia (FFI). The increasing levels of tau isoforms with exon-2 and exon-10 segments in the group of sCJD correlated well with the positive 14-3-3 in CSF. Additionally, the similar alterative profiles of tau isoforms with exon-2 and exon-10 segments were also observed in the brain tissues of scrapie-infected rodents and a sCJD patient. Our data here propose the tau isoforms with exon-2 and exon-10 segments increase in CSF of sCJD and some types of gCJD, which may help to understand the physiological metabolism and pathological significance of various tau isoforms in the pathogenesis of prion diseases. PMID:26188647

  14. NextSearch: A Search Engine for Mass Spectrometry Data against a Compact Nucleotide Exon Graph.

    PubMed

    Kim, Hyunwoo; Park, Heejin; Paek, Eunok

    2015-07-01

    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. PMID:26004133

  15. Loss of Methylation at GNAS Exon A/B Is Associated With Increased Intrauterine Growth

    PubMed Central

    Bréhin, Anne-Claire; Colson, Cindy; Maupetit-Méhouas, Stéphanie; Grybek, Virginie; Richard, Nicolas; Linglart, Agnès; Kottler, Marie-Laure

    2015-01-01

    Context: GNAS is one of few genetic loci that undergo allelic-specific methylation resulting in the parent-specific expression of at least four different transcripts. Due to monoallelic expression, heterozygous GNAS mutations affecting either paternally or maternally derived transcripts cause different forms of pseudohypoparathyroidism (PHP), including autosomal-dominant PHP type Ib (AD-PHP1B) associated with loss of methylation (LOM) at exon A/B alone or sporadic PHP1B (sporPHP1B) associated with broad GNAS methylation changes. Similar to effects other imprinted genes have on early development, we recently observed severe intrauterine growth retardation in newborns, later diagnosed with pseudopseudohypoparathyroidism (PPHP) because of paternal GNAS loss-of-function mutations. Objectives: This study aimed to determine whether GNAS methylation abnormalities affect intrauterine growth. Patients and Methods: Birth parameters were collected of patients who later developed sporPHP1B or AD-PHP1B, and of their healthy siblings. Comparisons were made to newborns affected by PPHP or PHP1A. Results: As newborns, AD-PHP1B patients were bigger than their healthy siblings and well above the reference average; increased sizes were particularly evident if the mothers were unaffected carriers of STX16 deletions. SporPHP1B newborns were slightly above average for weight and length, but their overgrowth was less pronounced than that of AD-PHP1B newborns from unaffected mothers. Conclusion: LOM at GNAS exon A/B due to maternal STX16 deletions and the resulting biallelic A/B expression are associated with enhanced fetal growth. These findings are distinctly different from those of PPHP patients with paternal GNAS exons 2–13 mutations, whose birth parameters are almost 4.5 z-scores below those of AD-PHP1B patients born to healthy mothers. PMID:25603460

  16. Identification of a novel exonic mutation at -13 from 5' splice site causing exon skipping in a girl with mitochondrial acetoacetyl-coenzyme A thiolase deficiency.

    PubMed Central

    Fukao, T; Yamaguchi, S; Wakazono, A; Orii, T; Hoganson, G; Hashimoto, T

    1994-01-01

    We identified a novel exonic mutation which causes exon skipping in the mitochondrial acetoacetyl-CoA thiolase (T2) gene from a girl with T2 deficiency (GK07). GK07 is a compound heterozygote; the maternal allele has a novel G to T transversion at position 1136 causing Gly379 to Val substitution (G379V) of the T2 precursor. In case of in vivo expression analysis, cells transfected with this mutant cDNA showed no evidence of restored T2 activity. The paternal allele was associated with exon 8 skipping at the cDNA level. At the gene level, a C to T transition causing Gln272 to termination codon (Q272STOP) was identified within exon 8, 13 bp from the 5' splice site of intron 8 in the paternal allele. The mRNA with Q272STOP could not be detected in GK07 fibroblasts, presumably because pre-mRNA with Q272STOP was unstable because of the premature termination. In vivo splicing experiments revealed that the exonic mutation caused partial skipping of exon 8. This substitution was thought to alter the secondary structure of T2 pre-mRNA around exon 8 and thus impede normal splicing. The role of exon sequences in the splicing mechanism is indicated by the exon skipping which occurred with an exonic mutation. Images PMID:7907600

  17. Selection preserves Ubiquitin Specific Protease 4 alternative exon skipping in therian mammals

    PubMed Central

    Vlasschaert, Caitlyn; Xia, Xuhua; Gray, Douglas A.

    2016-01-01

    Ubiquitin specific protease 4 (USP4) is a highly networked deubiquitinating enzyme with reported roles in cancer, innate immunity and RNA splicing. In mammals it has two dominant isoforms arising from inclusion or skipping of exon 7 (E7). We evaluated two plausible mechanisms for the generation of these isoforms: (A) E7 skipping due to a long upstream intron and (B) E7 skipping due to inefficient 5′ splice sites (5′SS) and/or branchpoint sites (BPS). We then assessed whether E7 alternative splicing is maintained by selective pressure or arose from genetic drift. Both transcript variants were generated from a USP4-E7 minigene construct with short flanking introns, an observation consistent with the second mechanism whereby differential splice signal strengths are the basis of E7 skipping. Optimization of the downstream 5′SS eliminated E7 skipping. Experimental validation of the correlation between 5′SS identity and exon skipping in vertebrates pinpointed the +6 site as the key splicing determinant. Therian mammals invariably display a 5′SS configuration favouring alternative splicing and the resulting isoforms have distinct subcellular localizations. We conclude that alternative splicing of mammalian USP4 is under selective maintenance and that long and short USP4 isoforms may target substrates in various cellular compartments. PMID:26833277

  18. A Duchenne Muscular Dystrophy Gene Hot Spot Mutation in Dystrophin-Deficient Cavalier King Charles Spaniels Is Amenable to Exon 51 Skipping

    PubMed Central

    Walmsley, Gemma L.; Arechavala-Gomeza, Virginia; Fernandez-Fuente, Marta; Burke, Margaret M.; Nagel, Nicole; Holder, Angela; Stanley, Rachael; Chandler, Kate; Marks, Stanley L.; Muntoni, Francesco; Shelton, G. Diane; Piercy, Richard J.

    2010-01-01

    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. PMID:20072625

  19. Harvey ras genes transform without mutant codons, apparently activated by truncation of a 5' exon (exon -1).

    PubMed Central

    Cichutek, K; Duesberg, P H

    1986-01-01

    The hypothesis is tested that the ras gene of Harvey sarcoma virus (Ha-SV) and the proto-ras DNAs from certain tumor cells derive transforming function from specific codons in which they differ from normal proto-ras genes. Molecularly cloned Harvey proviral vectors carrying viral ras, normal rat proto-ras, and recombinant ras genes in which the virus-specific ras codons 12 and 59 were replaced by proto-ras equivalents each transformed aneuploid mouse 3T3 cells after latent periods that ranged from 4 to 10 days. Viruses with or without virus-specific ras codons all transformed diploid rat cells in 3-5 days equally well. However, in the absence of virus replication, mutant codons were beneficial for transforming function. Deletion of non-ras regions of Ha-SV did not affect transforming function. We conclude that specific ras codons are not necessary for transforming function. Comparisons of the ras sequences of Ha-SV, BALB SV, and Rasheed SV with sequences of proto-ras genes from rat and man revealed an upstream proto-ras exon, termed exon -1. The 3' end of this exon is present in all three viruses and in a ras pseudogene of the rat. Since ras genes transform without mutation and since exon -1 is truncated in viral ras genes and all transforming proto-ras DNAs of the Harvey and the Kirsten ras family, we propose that ras genes are activated by truncation of exon -1 either via viral transduction or artificially via cloning and transfection. The proposal implies that untruncated proto-ras genes with point mutations may not be cellular cancer genes. Images PMID:3517865

  20. Structural basis for exon recognition by a group II intron

    SciTech Connect

    Toor, Navtej; Rajashankar, Kanagalaghatta; Keating, Kevin S.; Pyle, Anna Marie

    2008-11-18

    Free group II introns are infectious retroelements that can bind and insert themselves into RNA and DNA molecules via reverse splicing. Here we report the 3.4-A crystal structure of a complex between an oligonucleotide target substrate and a group IIC intron, as well as the refined free intron structure. The structure of the complex reveals the conformation of motifs involved in exon recognition by group II introns.

  1. Exon-intron organization and sequence comparison of human and murine T11 (CD2) genes

    SciTech Connect

    Diamond, D.J.; Clayton, L.K.; Sayre, P.H.; Reinherz, E.L.

    1988-03-01

    Genomic DNA clones containing the human and murine genes coding for the 50-kDa T11 (CD2) T-cell surface glycoprotein were characterized. The human T11 gene is approx. = 12 kilobases long and comprised of five exons. A leader exon (L) contains the 5'-untranslated region and most of the nucleotides defining the signal peptide (amino acids (aa) -24 to -5). Two exons encode the extracellular segment; exon Ex1 is 321 base pairs (bp) long and codes for four residues of the leader peptide and aa 1-103 of the mature protein, and exon Ex2 is 231 bp long and encodes aa 104-180. Exon TM is 123 bp long and codes for the single transmembrane region of the molecule (aa 181-221). Exon C is a large 765-bp exon encoding virtually the entire cytoplasmic domain (aa 222-327) and the 3'-untranslated region. The murine region T11 gene has a similar organization with exon-intron boundaries essentially identical to the human gene. Substantial conservation of nucleotide sequences between species in both 5'- and 3'-gene flanking regions equivalent to that among homologous exons suggests that murine and human genes may be regulated in a similar fashion. The probable relationship of the individual T11 exons to functional and structural protein domains is discussed.

  2. Developmental and muscle-type-specific expression of mouse nebulin exons 127 and 128.

    PubMed

    Donner, Kati; Nowak, Kristen J; Aro, Mimmi; Pelin, Katarina; Wallgren-Pettersson, Carina

    2006-10-01

    The human nebulin gene includes 183 exons and four regions of alternative splicing. The mouse nebulin gene, with 166 exons, has a similar organization. Here we describe the expression patterns of one of the alternatively spliced regions of nebulin: exons 127 and 128 in the mouse gene, corresponding to human nebulin exons 143 and 144. Expression was elucidated by quantifying the differentially spliced transcripts in mice of different ages. In most of the muscles studied, transcripts expressing exon 127 were more prominent in muscles from younger mice, while older mice showed higher quantities of the transcript expressing exon 128. Some muscles, e.g., diaphragm and masseter, almost exclusively expressed only one of the two transcripts, whereas others, e.g., soleus and cardiac muscle, expressed equal quantities of both transcripts. The expression patterns did not correlate with fiber-type composition. We speculate that these exons harbor a regulatory function utilized during muscle maturation. PMID:16860535

  3. A novel bipartite splicing enhancer modulates the differential processing of the human fibronectin EDA exon.

    PubMed Central

    Caputi, M; Casari, G; Guenzi, S; Tagliabue, R; Sidoli, A; Melo, C A; Baralle, F E

    1994-01-01

    EDA is a facultative type III homology of human fibronectin encoded by an alternative spliced exon. The EDA+ and EDA- mRNA forms show a cell type specific distribution with their relative proportion varying during development, aging and oncogenic transformation. We have previously demonstrated that an 81 bp nucleotide sequence within the exon itself is essential for differential RNA processing. Fine mapping of cis acting elements within this region has been carried out to identify possible target sites for the modulation of alternative splicing. There are at least two short nucleotide sequences involved. Element A (GAAGAAGA) is a positive modulator for the recognition of the exon, its deletion results in constitutive exclusion of the EDA exon. Element B (CAAGG) is a negative modulator for exon recognition, its deletion results in constitutive inclusion of the EDA exon. This bipartite structure of the splicing enhancer is a novel feature of the mammalian exons. Images PMID:8152907

  4. Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia.

    PubMed

    Gray, Mary J; Kannu, Peter; Sharma, Swarkar; Neyt, Christine; Zhang, Dongping; Paria, Nandina; Daniel, Philip B; Whetstone, Heather; Sprenger, Hans-Georg; Hammerschmidt, Philipp; Weng, Angela; Dupuis, Lucie; Jobling, Rebekah; Mendoza-Londono, Roberto; Dray, Michael; Su, Peiqiang; Wilson, Megan J; Kapur, Raj P; McCarthy, Edward F; Alman, Benjamin A; Howard, Andrew; Somers, Gino R; Marshall, Christian R; Manners, Simon; Flanagan, Adrienne M; Rathjen, Karl E; Karol, Lori A; Crawford, Haemish; Markie, David M; Rios, Jonathan J; Wise, Carol A; Robertson, Stephen P

    2015-12-01

    The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (MET(Δ14)) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the MET(Δ14) mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone. PMID:26637977

  5. Human decorin gene: Intron-exon junctions and chromosomal localization

    SciTech Connect

    Vetter, U.; Young, M.F.; Fisher, L.W. ); Vogel, W.; Just, W. )

    1993-01-01

    All of the protein-encoding exons and the 3[prime]flanking region of the human decorin gene have been cloned an partially sequenced. The locations of the intron-exon junctions within the coding portion of the gene were identical to those found for the homologous human gene, biglycan. The sizes of the introns in the decorin gene, however, were substantially larger than those of the same introns of the biglycan gene. Portions of introns 1, 2, and 3 as well as exon 1 were not found during our extensive screening process. The 5[prime] end of intron 2 was found to have an AG-rich region followed immediately by a CT-rich region. Furthermore, the 5[prime] end of intron 3 was very rich in thymidine, whereas the 3[prime] end of intron 7 was rich in adenosine. Several cDNA clones constructed from cultured human bone cell mRNA were found to contain a different sequence at the 5[prime] end compared to that previously published for mRNA from a human embryonic fibroblast cell line. We were also unable to find the alternate 3[prime] flanking region of the previously published cDNA sequence. We have mapped the human decorin gene by in situ methods to chromosome 12q2l.3. 30 refs., 3 figs., 1 tab.

  6. Mutations Preventing Regulated Exon Skipping in MET Cause Osteofibrous Dysplasia

    PubMed Central

    Gray, Mary J.; Kannu, Peter; Sharma, Swarkar; Neyt, Christine; Zhang, Dongping; Paria, Nandina; Daniel, Philip B.; Whetstone, Heather; Sprenger, Hans-Georg; Hammerschmidt, Philipp; Weng, Angela; Dupuis, Lucie; Jobling, Rebekah; Mendoza-Londono, Roberto; Dray, Michael; Su, Peiqiang; Wilson, Megan J.; Kapur, Raj P.; McCarthy, Edward F.; Alman, Benjamin A.; Howard, Andrew; Somers, Gino R.; Marshall, Christian R.; Manners, Simon; Flanagan, Adrienne M.; Rathjen, Karl E.; Karol, Lori A.; Crawford, Haemish; Markie, David M.; Rios, Jonathan J.; Wise, Carol A.; Robertson, Stephen P.

    2015-01-01

    The periosteum contributes to bone repair and maintenance of cortical bone mass. In contrast to the understanding of bone development within the epiphyseal growth plate, factors that regulate periosteal osteogenesis have not been studied as intensively. Osteofibrous dysplasia (OFD) is a congenital disorder of osteogenesis and is typically sporadic and characterized by radiolucent lesions affecting the cortical bone immediately under the periosteum of the tibia and fibula. We identified germline mutations in MET, encoding a receptor tyrosine kinase, that segregate with an autosomal-dominant form of OFD in three families and a mutation in a fourth affected subject from a simplex family and with bilateral disease. Mutations identified in all families with dominant inheritance and in the one simplex subject with bilateral disease abolished the splice inclusion of exon 14 in MET transcripts, which resulted in a MET receptor (METΔ14) lacking a cytoplasmic juxtamembrane domain. Splice exclusion of this domain occurs during normal embryonic development, and forced induction of this exon-exclusion event retarded osteoblastic differentiation in vitro and inhibited bone-matrix mineralization. In an additional subject with unilateral OFD, we identified a somatic MET mutation, also affecting exon 14, that substituted a tyrosine residue critical for MET receptor turnover and, as in the case of the METΔ14 mutations, had a stabilizing effect on the mature protein. Taken together, these data show that aberrant MET regulation via the juxtamembrane domain subverts core MET receptor functions that regulate osteogenesis within cortical diaphyseal bone. PMID:26637977

  7. Translational and regulatory challenges for exon skipping therapies.

    PubMed

    Aartsma-Rus, Annemieke; Ferlini, Alessandra; Goemans, Nathalie; Pasmooij, Anna M G; Wells, Dominic J; Bushby, Katerine; Vroom, Elizabeth; Balabanov, Pavel

    2014-10-01

    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 interpretation of appropriate clinical outcome measures. Others are inherent to the antisense oligonucleotide (AON)-mediated exon skipping approach, which employs small modified DNA or RNA molecules to manipulate the splicing process. This is a new approach and only limited information is available on long-term safety and toxicity for most AON chemistries. Furthermore, AONs often act in a mutation-specific manner, in which case multiple AONs have to be developed for a single disease. A workshop focusing on preclinical development, trial design, outcome measures, and different forms of marketing authorization was organized by the regulatory models and biochemical outcome measures working groups of Cooperation of Science and Technology Action: "Networking towards clinical application of antisense-mediated exon skipping for rare diseases." The workshop included participants from patient organizations, academia, and members of staff from the European Medicine Agency and Medicine Evaluation Board (the Netherlands). This statement article contains the key outcomes of this meeting. PMID:25184444

  8. Antisense modulation of both exonic and intronic splicing motifs induces skipping of a DMD pseudo-exon responsible for x-linked dilated cardiomyopathy.

    PubMed

    Rimessi, Paola; Fabris, Marina; Bovolenta, Matteo; Bassi, Elena; Falzarano, Sofia; Gualandi, Francesca; Rapezzi, Claudio; Coccolo, Fabio; Perrone, Daniela; Medici, Alessandro; Ferlini, Alessandra

    2010-09-01

    Antisense-mediated exon skipping has proven to be efficacious for subsets of Duchenne muscular dystrophy mutations. This approach is based on targeting specific splicing motifs that interfere with the spliceosome assembly by steric hindrance. Proper exon recognition by the splicing machinery is thought to depend on exonic splicing enhancer sequences, often characterized by purine-rich stretches, representing potential targets for antisense-mediated exon skipping. We identified and functionally characterized two purine-rich regions located within dystrophin intron 11 and involved in splicing regulation of a pseudo-exon. A functional role for these sequences was suggested by a pure intronic DMD deletion causing X-linked dilated cardiomyopathy through the prevalent cardiac incorporation of the aberrant pseudo-exon, marked as Alu-exon, into the dystrophin transcript. The first splicing sequence is contained within the pseudo-exon, whereas the second is localized within its 3' intron. We demonstrated that the two sequences actually behave as splicing enhancers in cell-free splicing assays because their deletion strongly interferes with the pseudo-exon inclusion. Cell-free results were then confirmed in myogenic cells derived from the patient with X-linked dilated cardiomyopathy, by targeting the identified motifs with antisense molecules and obtaining a reduction in dystrophin pseudo-exon recognition. The splicing motifs identified could represent target sequences for a personalized molecular therapy in this particular DMD mutation. Our results demonstrated for the first time the role of intronic splicing sequences in antisense modulation with implications in exon skipping-mediated therapeutic approaches. PMID:20486769

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

    PubMed

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

    2016-01-01

    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. PMID:27285612

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

    PubMed Central

    Kuraoka, Mutsuki; Lee, Joshua J.A.; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    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. PMID:27285612

  11. A synonymous mutation in SPINK5 exon 11 causes Netherton syndrome by altering exonic splicing regulatory elements.

    PubMed

    Fortugno, Paola; Grosso, Fabiana; Zambruno, Giovanna; Pastore, Serena; Faletra, Flavio; Castiglia, Daniele

    2012-05-01

    Netherton syndrome (NS) is a rare, life-threatening ichthyosiform syndrome caused by recessive loss-of-function mutations in SPINK5 gene encoding lymphoepithelial Kazal-type-related inhibitor (LEKTI), a serine protease inhibitor expressed in the most differentiated epidermal layers and crucial for skin barrier function. We report the functional characterization of a previously unrecognized synonymous variant, c.891C>T (p.Cys297Cys), identified in the SPINK5 exon 11 of an NS patient. We demonstrated that the c.891C>T mutation is associated with abnormal pre-mRNA splicing and residual LEKTI expression in the patient's keratinocytes. Subsequent minigene splicing assays and in silico predictions confirmed the direct role of the synonymous mutation in inhibiting exon 11 inclusion by a mechanism that involves the activity of exonic regulatory sequences, namely splicing enhancer and silencer. However, this deleterious effect was not complete and a residual amount of normal mRNA and LEKTI protein could be detected, correlating with the relatively mild patient's phenotype. Our study represents the first identification of a disease-causing SPINK5 mutation that alters splicing without affecting canonical splice sites. PMID:22377713

  12. AVCRI104P3, a novel multitarget compound with cognition-enhancing and anxiolytic activities: studies in cognitively poor middle-aged mice.

    PubMed

    Giménez-Llort, L; Ratia, M; Pérez, B; Camps, P; Muñoz-Torrero, D; Badia, A; Clos, M V

    2015-06-01

    The present work describes, for the first time, the in vivo effects of the multitarget compound AVCRI104P3, a new anticholinesterasic drug with potent inhibitory effects on human AChE, human BuChE and BACE-1 activities as well as on the AChE-induced and self-induced Aβ aggregation. We characterized the behavioral effects of chronic treatment with AVCRI104P3 (0.6 μmol kg(-1), i.p., 21 days) in a sample of middle aged (12-month-old) male 129/Sv×C57BL/6 mice with poor cognitive performance, as shown by the slow acquisition curves of saline-treated animals. Besides, a comparative assessment of cognitive and non-cognitive actions was done using its in vitro equipotent doses of huprine X (0.12 μmol kg(-1)), a huperzine A-tacrine hybrid. The screening assessed locomotor activity, anxiety-like behaviors, cognitive function and side effects. The results on the 'acquisition' of spatial learning and memory show that AVCRI104P3 exerted pro-cognitive effects improving both short- and long-term processes, resulting in a fast and efficient acquisition of the place task in the Morris water maze. On the other hand, a removal test and a perceptual visual learning task indicated that both AChEIs improved short-term 'memory' as compared to saline treated mice. Both drugs elicited the same response in the corner test, but only AVCRI104P3 exhibited anxiolytic-like actions in the dark/light box test. These cognitive-enhancement and anxiolytic-like effects demostrated herein using a sample of middle-aged animals and the lack of adverse effects, strongly encourage further studies on AVCRI104P3 as a promising multitarget therapeutic agent for the treatment of cholinergic dysfunction underlying natural aging and/or dementias. PMID:25732954

  13. Antifungal Mechanism of Action of Lactoferrin: Identification of H+-ATPase (P3A-Type) as a New Apoptotic-Cell Membrane Receptor.

    PubMed

    Andrés, María T; Acosta-Zaldívar, Maikel; Fierro, José F

    2016-07-01

    Human lactoferrin (hLf) is a protein of the innate immune system which induces an apoptotic-like process in yeast. Determination of the susceptibility to lactoferrin of several yeast species under different metabolic conditions, respiratory activity, cytoplasmic ATP levels, and external medium acidification mediated by glucose assays suggested plasma membrane Pma1p (P3A-type ATPase) as the hLf molecular target. The inhibition of plasma membrane ATPase activity by hLf and the identification of Pma1p as the hLf-binding membrane protein confirmed the previous physiological evidence. Consistent with this, cytoplasmic ATP levels progressively increased in hLf-treated Candida albicans cells. However, oligomycin, a specific inhibitor of the mitochondrial F-type ATPase proton pump (mtATPase), abrogated the antifungal activity of hLf, indicating a crucial role for mtATPase in the apoptotic process. We suggest that lactoferrin targeted plasma membrane Pma1p H(+)-ATPase, perturbing the cytoplasmic ion homeostasis (i.e., cytoplasmic H(+) accumulation and subsequent K(+) efflux) and inducing a lethal mitochondrial dysfunction. This initial event involved a normal mitochondrial ATP synthase activity responsible for both the ATP increment and subsequent hypothetical mitochondrial proton flooding process. We conclude that human lactoferrin inhibited Pma1p H(+)-ATPase, inducing an apoptotic-like process in metabolically active yeast. Involvement of mitochondrial H(+)-ATPase (nonreverted) was essential for the progress of this programmed cell death in which the ionic homeostasis perturbation seems to precede classical nonionic apoptotic events. PMID:27139463

  14. Absence of an Intron Splicing Silencer in Porcine Smn1 Intron 7 Confers Immunity to the Exon Skipping Mutation in Human SMN2

    PubMed Central

    Doktor, Thomas Koed; Schrøder, Lisbeth Dahl; Andersen, Henriette Skovgaard; Brøner, Sabrina; Kitewska, Anna; Sørensen, Charlotte Brandt; Andresen, Brage Storstein

    2014-01-01

    Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole. PMID:24892836

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

    PubMed Central

    Findlay, Andrew R.; Wein, Nicolas; Kaminoh, Yuuki; Taylor, Laura E.; Dunn, Diane M.; Mendell, Jerry R.; King, Wendy M.; Pestronk, Alan; Florence, Julaine M.; Mathews, Katherine D.; Finkel, Richard S.; Swoboda, Kathryn J.; Howard, Michael T.; Day, John W.; McDonald, Craig; Nicolas, Aurélie; Le Rumeur, Elisabeth; Weiss, Robert B.; Flanigan, Kevin M.

    2015-01-01

    Objective Exon-skipping therapies aim to convert Duchenne muscular dystrophy (DMD) into less severe Becker muscular dystrophy (BMD) by altering pre-mRNA splicing to restore an open reading frame, allowing translation of an internally deleted and partially functional dystrophin protein. The most common single exon deletion – exon 45 (Δ45) – may theoretically be treated by skipping of either flanking exon (44 or 46). We sought to predict the impact of these by assessing the clinical severity in dystrophinopathy patients. Methods Phenotypic data including clinical diagnosis, age at wheelchair use, age at loss of ambulation, and presence of cardiomyopathy was analyzed from 41 dystrophinopathy patients containing equivalent in-frame deletions. Results As expected, deletions of either exons 45-47 (Δ45–47) or exons 45-48 (Δ45-48) result in BMD in 97% (36/37) of subjects. Unexpectedly, deletion of exons 45-46 (Δ45-46) is associated with the more severe DMD phenotype in 4/4 subjects despite an in-frame transcript. Notably, no patients with a deletion of exons 44-45 (Δ44-45) were found within the UDP database, and this mutation has only been reported twice before, which suggests an ascertainment bias attributable to a very mild phenotype. Interpretation The observation that Δ45-46 patients have typical DMD suggests that the conformation of the resultant protein may result in protein instability or altered binding of critical partners. We conclude that in DMD patients with Δ45, skipping of either exon 44 or multi-exon skipping of exons 46 and 47 (or exons 46-48) are better potential therapies than skipping of exon 46 alone. PMID:25612243

  16. CRIPT exonic deletion and a novel missense mutation in a female with short stature, dysmorphic features, microcephaly, and pigmentary abnormalities.

    PubMed

    Leduc, Magalie S; Niu, Zhiyv; Bi, Weimin; Zhu, Wenmiao; Miloslavskaya, Irene; Chiang, Theodore; Streff, Haley; Seavitt, John R; Murray, Stephen A; Eng, Christine; Chan, Audrey; Yang, Yaping; Lalani, Seema R

    2016-08-01

    Mutations in CRIPT encoding cysteine-rich PDZ domain-binding protein are rare, and to date have been reported in only two patients with autosomal recessive primordial dwarfism and distinctive facies. Here, we describe a female with biallelic mutations in CRIPT presenting with postnatal growth retardation, global developmental delay, and dysmorphic features including frontal bossing, high forehead, and sparse hair and eyebrows. Additional clinical features included high myopia, admixed hyper- and hypopigmented macules primarily on the face, arms, and legs, and syndactyly of 4-5 toes bilaterally. Using whole exome sequencing (WES) and chromosomal microarray analysis (CMA), we detected a c.8G>A (p.C3Y) missense variant in exon 1 of the CRIPT gene inherited from the mother and a 1,331 bp deletion encompassing exon 1, inherited from the father. The c.8G>A (p.C3Y) missense variant in CRIPT was apparently homozygous in the proband due to the exon 1 deletion. Our findings illustrate the clinical utility of combining WES with copy number variant (CNV) analysis to provide a molecular diagnosis to patients with rare Mendelian disorders. Our findings also illustrate the clinical spectrum of CRIPT related mutations. © 2016 Wiley Periodicals, Inc. PMID:27250922

  17. Biochemical identification of new proteins involved in splicing repression at the Drosophila P-element exonic splicing silencer

    PubMed Central

    Horan, Lucas; Yasuhara, Jiro C.; Kohlstaedt, Lori A.; Rio, Donald C.

    2015-01-01

    Splicing of the Drosophila P-element third intron (IVS3) is repressed in somatic tissues due to the function of an exonic splicing silencer (ESS) complex present on the 5′ exon RNA. To comprehensively characterize the mechanisms of this alternative splicing regulation, we used biochemical fractionation and affinity purification to isolate the silencer complex assembled in vitro and identify the constituent proteins by mass spectrometry. Functional assays using splicing reporter minigenes identified the proteins hrp36 and hrp38 and the cytoplasmic poly(A)-binding protein PABPC1 as novel functional components of the splicing silencer. hrp48, PSI, and PABPC1 have high-affinity RNA-binding sites on the P-element IVS3 5′ exon, whereas hrp36 and hrp38 proteins bind with low affinity to the P-element silencer RNA. RNA pull-down and immobilized protein assays showed that hrp48 protein binding to the silencer RNA can recruit hrp36 and hrp38. These studies identified additional components that function at the P-element ESS and indicated that proteins with low-affinity RNA-binding sites can be recruited in a functional manner through interactions with a protein bound to RNA at a high-affinity binding site. These studies have implications for the role of heterogeneous nuclear ribonucleoproteins (hnRNPs) in the control of alternative splicing at cis-acting regulatory sites. PMID:26545814

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

    PubMed

    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 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. PMID:19495418

  19. Characterization of two exon-skipping mutations (3120G{r_arrow}A, 3600G{r_arrow}A) in the CFTR gene

    SciTech Connect

    Zielendki, J.; Markiewicz, D.; Ainsworth, P.J.

    1994-09-01

    Many different types of mutations have been identified in the CFTR gene in patients with cystic fibrosis. Due to the large size of the gene (230 kb), CF mutations have been primarily detected by genomic DNA analysis. While some of the sequence alterations, such as nonsense and frameshift mutations, provide immediate clues to possible molecular consequence, others such as missense mutations are less apparent in their involvement in the disease. In our systematic scanning of the entire coding regions of the CFTR gene for a group of CF patients carrying unknown mutations, two different G to A substitutions located at the last nucleotide position of an exon were identified in two patients. The first one, 3120G{r_arrow}A, is located in exon 16 and the other one, 3600G{r_arrow}A, in exon 18 of the CFTR gene. Both of them are also located at the third position of the corresponding amino acid codon (CAG and TTC, respectively). As a result, the changes would not affect the encoded amino acids (Glu and Leu, respectively). To demonstrate that these are in fact pathologic mutations, we have investigated the CFTR transcripts in these two patients. The results of RT-PCR analysis revealed that aberrant splicing occurred in both cases: transcripts missing exon 16 and 18 were present in the 2 patients, respectively. No normal product was detectable from the 3120G{r_arrow}A and 3600G{r_arrow}A alleles, suggesting that the normal-sized products were exclusively derived from the {triangle}F508 mutant alleles in both of these patients. Hence, we conclude that both 3120G{r_arrow}A and 3600G{r_arrow}A mutations cause exon-skipping leading to premature termination and truncation of CFTR and that the altered G residue in each of these exons is probably part of the splice donor sequence important for efficient mRNA splicing.

  20. Novel methodologies for spectral classification of exon and intron sequences

    NASA Astrophysics Data System (ADS)

    Kwan, Hon Keung; Kwan, Benjamin Y. M.; Kwan, Jennifer Y. Y.

    2012-12-01

    Digital processing of a nucleotide sequence requires it to be mapped to a numerical sequence in which the choice of nucleotide to numeric mapping affects how well its biological properties can be preserved and reflected from nucleotide domain to numerical domain. Digital spectral analysis of nucleotide sequences unfolds a period-3 power spectral value which is more prominent in an exon sequence as compared to that of an intron sequence. The success of a period-3 based exon and intron classification depends on the choice of a threshold value. The main purposes of this article are to introduce novel codes for 1-sequence numerical representations for spectral analysis and compare them to existing codes to determine appropriate representation, and to introduce novel thresholding methods for more accurate period-3 based exon and intron classification of an unknown sequence. The main findings of this study are summarized as follows: Among sixteen 1-sequence numerical representations, the K-Quaternary Code I offers an attractive performance. A windowed 1-sequence numerical representation (with window length of 9, 15, and 24 bases) offers a possible speed gain over non-windowed 4-sequence Voss representation which increases as sequence length increases. A winner threshold value (chosen from the best among two defined threshold values and one other threshold value) offers a top precision for classifying an unknown sequence of specified fixed lengths. An interpolated winner threshold value applicable to an unknown and arbitrary length sequence can be estimated from the winner threshold values of fixed length sequences with a comparable performance. In general, precision increases as sequence length increases. The study contributes an effective spectral analysis of nucleotide sequences to better reveal embedded properties, and has potential applications in improved genome annotation.

  1. SRSF2 Mutations Contribute to Myelodysplasia Through Mutant-Specific Effects on Exon Recognition

    PubMed Central

    Kim, Eunhee; Ilagan, Janine O.; Liang, Yang; Daubner, Gerrit M.; Lee, Stanley C.-W.; Ramakrishnan, Aravind; Li, Yue; Chung, Young Rock; Micol, Jean-Baptiste; Murphy, Michele; Cho, Hana; Hana, Min-Kyung; Zebari, Ahmad S.; Aumann, Shlomzion; Park, Christopher Y.; Buonamici, Silvia; Smith, Peter G.; Deeg, H. Joachim; Lobry, Camille; Aifantis, Iannis; Modis, Yorgo; Allain, Frederic H.-T.; Halene, Stephanie; Bradley, Robert K.; Abdel-Wahab, Omar

    2015-01-01

    SUMMARY Mutations affecting spliceosomal proteins are the most common class of mutations in patients with myelodysplastic syndromes (MDS), yet their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2’s normal sequence-specific RNA binding activity, thereby altering recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2 that triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in splicing of key regulators, and impaired hematopoiesis. PMID:25965569

  2. SRSF2 Mutations Contribute to Myelodysplasia by Mutant-Specific Effects on Exon Recognition.

    PubMed

    Kim, Eunhee; Ilagan, Janine O; Liang, Yang; Daubner, Gerrit M; Lee, Stanley C-W; Ramakrishnan, Aravind; Li, Yue; Chung, Young Rock; Micol, Jean-Baptiste; Murphy, Michele E; Cho, Hana; Kim, Min-Kyung; Zebari, Ahmad S; Aumann, Shlomzion; Park, Christopher Y; Buonamici, Silvia; Smith, Peter G; Deeg, H Joachim; Lobry, Camille; Aifantis, Iannis; Modis, Yorgo; Allain, Frederic H-T; Halene, Stephanie; Bradley, Robert K; Abdel-Wahab, Omar

    2015-05-11

    Mutations affecting spliceosomal proteins are the most common mutations in patients with myelodysplastic syndromes (MDS), but their role in MDS pathogenesis has not been delineated. Here we report that mutations affecting the splicing factor SRSF2 directly impair hematopoietic differentiation in vivo, which is not due to SRSF2 loss of function. By contrast, SRSF2 mutations alter SRSF2's normal sequence-specific RNA binding activity, thereby altering the recognition of specific exonic splicing enhancer motifs to drive recurrent mis-splicing of key hematopoietic regulators. This includes SRSF2 mutation-dependent splicing of EZH2, which triggers nonsense-mediated decay, which, in turn, results in impaired hematopoietic differentiation. These data provide a mechanistic link between a mutant spliceosomal protein, alterations in the splicing of key regulators, and impaired hematopoiesis. PMID:25965569

  3. The complete local genotype–phenotype landscape for the alternative splicing of a human exon

    PubMed Central

    Julien, Philippe; Miñana, Belén; Baeza-Centurion, Pablo; Valcárcel, Juan; Lehner, Ben

    2016-01-01

    The properties of genotype–phenotype landscapes are crucial for understanding evolution but are not characterized for most traits. Here, we present a >95% complete local landscape for a defined molecular function—the alternative splicing of a human exon (FAS/CD95 exon 6, involved in the control of apoptosis). The landscape provides important mechanistic insights, revealing that regulatory information is dispersed throughout nearly every nucleotide in an exon, that the exon is more robust to the effects of mutations than its immediate neighbours in genotype space, and that high mutation sensitivity (evolvability) will drive the rapid divergence of alternative splicing between species unless it is constrained by selection. Moreover, the extensive epistasis in the landscape predicts that exonic regulatory sequences may diverge between species even when exon inclusion levels are functionally important and conserved by selection. PMID:27161764

  4. The complete local genotype-phenotype landscape for the alternative splicing of a human exon.

    PubMed

    Julien, Philippe; Miñana, Belén; Baeza-Centurion, Pablo; Valcárcel, Juan; Lehner, Ben

    2016-01-01

    The properties of genotype-phenotype landscapes are crucial for understanding evolution but are not characterized for most traits. Here, we present a >95% complete local landscape for a defined molecular function-the alternative splicing of a human exon (FAS/CD95 exon 6, involved in the control of apoptosis). The landscape provides important mechanistic insights, revealing that regulatory information is dispersed throughout nearly every nucleotide in an exon, that the exon is more robust to the effects of mutations than its immediate neighbours in genotype space, and that high mutation sensitivity (evolvability) will drive the rapid divergence of alternative splicing between species unless it is constrained by selection. Moreover, the extensive epistasis in the landscape predicts that exonic regulatory sequences may diverge between species even when exon inclusion levels are functionally important and conserved by selection. PMID:27161764

  5. Conserved intron elements repress splicing of a neuron-specific c-src exon in vitro.

    PubMed Central

    Chan, R C; Black, D L

    1995-01-01

    The neuron-specific N1 exon of the mouse c-src transcript is normally skipped in nonneuronal cells. In this study, we examined the sequence requirements for the exclusion of this exon in nonneuronal HeLa cell nuclear extracts. We found that the repression of the N1 exon is mediated by specific intron sequences that flank the N1 exon. Mutagenesis experiments identified conserved CUCUCU elements within these intron regions that are required for the repression of N1 splicing. The addition of an RNA competitor containing the upstream regulatory sequence to the HeLa extract induced splicing of the intron downstream of N1, indicating that the competitor sequence binds to splicing repressor proteins. The similarities between this mechanism for src splicing repression and the repression of other regulated exons point to a common role of exon-spanning interactions in splicing repression. PMID:7565790

  6. TALE-directed local modulation of H3K9 methylation shapes exon recognition

    PubMed Central

    Bieberstein, Nicole I.; Kozáková, Eva; Huranová, Martina; Thakur, Prasoon K.; Krchňáková, Zuzana; Krausová, Michaela; Oesterreich, Fernando Carrillo; Staněk, David

    2016-01-01

    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. PMID:27439481

  7. TALE-directed local modulation of H3K9 methylation shapes exon recognition.

    PubMed

    Bieberstein, Nicole I; Kozáková, Eva; Huranová, Martina; Thakur, Prasoon K; Krchňáková, Zuzana; Krausová, Michaela; Carrillo Oesterreich, Fernando; Staněk, David

    2016-01-01

    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. PMID:27439481

  8. Hypomethylation of the interphotoreceptor retinoid-binding protein (IRBP) promotor and first exon is linked to expression of the gene.

    PubMed Central

    Albini, A; Toffenetti, J; Zhu, Z; Chader, G J; Noonan, D M

    1990-01-01

    The interphotoreceptor retinoid-binding protein (IRBP) is limited in expression to retinal photoreceptor cells and a subset of pinealocytes. We have obtained a genomic clone containing the entire coding region and 7 kb of 5' flanking sequence. As a first step in studying IRBP gene regulation we have examined the CpG methylation patterns of the entire IRBP gene in expressing and non-expressing human cells. This has been done by isolation of high molecular weight DNA from Y-79 cells grown in suspension or attached to poly-D-lysine, which synthesize IRBP at different levels, and from human lymphocytes, which were shown by northern analysis to lack IRBP message. The DNA was digested by either Hpa II, Msp I, or Hha I. Southern blots were prepared with these digests and hybridized with probes made from fragments covering the complete genomic clone. Probes from the first exon, the introns and the 3' end gave banding patterns which showed no differences between the expressing cells and the lymphocytes. A probe from the very 5' end did not give a clear banding pattern, probably due to the presence of repetitive elements in the probe. However, a Hind III probe covering the 5' flanking 3 kb and the beginning of the first exon hybridized with a 1.8 kb band in Hpa II digests of Y-79 cells which was not present in Hpa II digests of lymphocyte DNA. In addition, a 2.1-2.3 kb Hha I band was found only in the Y-79 DNA digests. Sequence analysis of the promoter region indicated that these bands were due to hypomethylation of sites within a CpG rich island from -1578 to -1108 in the promoter and hypomethylation of sites in the beginning of the first exon. A Hha I site between the CpG island and the first exon was not hypomethylated in the expressing Y-79 cells. We propose that hypomethylation of the CpG rich island of the IRBP promoter and the first exon is linked to the expression of this gene. Images PMID:2402443

  9. Biased exon/intron distribution of cryptic and de novo 3′ splice sites

    PubMed Central

    Královičová, Jana; Christensen, Mikkel B.; Vořechovský, 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 ∼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. PMID:16141195

  10. Multiple interdependent sequence elements control splicing of a fibroblast growth factor receptor 2 alternative exon.

    PubMed Central

    Del Gatto, F; Plet, A; Gesnel, M C; Fort, C; Breathnach, R

    1997-01-01

    The fibroblast growth factor receptor 2 gene contains a pair of mutually exclusive alternative exons, one of which (K-SAM) is spliced specifically in epithelial cells. We have described previously (F. Del Gatto and R. Breathnach, Mol. Cell. Biol. 15:4825-4834, 1995) some elements controlling K-SAM exon splicing, namely weak exon splice sites, an exon-repressing sequence, and an intron-activating sequence. We identify here two additional sequences in the intron downstream from the K-SAM exon which activate splicing of the exon. The first sequence (intron-activating sequence 2 [IAS2]) lies 168 to 186 nucleotides downstream from the exon's 5' splice site. The second sequence (intron-activating sequence 3 [IAS3]) lies 933 to 1,052 nucleotides downstream from the exon's 5' splice site. IAS3 is a complex region composed of several parts, one of which (nucleotides 963 to 983) can potentially form an RNA secondary structure with IAS2. This structure is composed of two stems separated by an asymmetric bulge. Mutations which disrupt either stem decrease activation, while compensatory mutations which reestablish the stem restore activation, either completely or partially, depending on the mutation. We present a model for K-SAM exon splicing involving the intervention of multiple, interdependent pre-mRNA sequence elements. PMID:9271388

  11. SOX11 MODULATES BRAIN-DERIVED NEUROTROPHIC FACTOR EXPRESSION IN AN EXON PROMOTER-SPECIFIC MANNER

    PubMed Central

    Salerno, Kathleen M.; Jing, Xiaotang; Diges, Charlotte M.; Cornuet, Pamela K.; Glorioso, Joseph C.; Albers, Kathryn M.

    2011-01-01

    Sox11 is a high mobility group (HMG) containing transcription factor that is significantly elevated in peripheral neurons in response to nerve injury. In vitro and in vivo studies support a central role for Sox11 in adult neuron growth and survival following injury. Brain-derived neurotrophic factor (BDNF) is a pleiotropic growth factor that has effects on neuronal survival, differentiation, synaptic plasticity and regeneration. BDNF transcription is elevated in the DRG following nerve injury in parallel with Sox11 allowing for the possible regulation by Sox11. To begin to assess the possible influence of Sox11 we used reverse transcriptase PCR assays to determine the relative expression of the nine (I-IXa) noncoding exons and one coding exon (exon IX) of the BDNF gene after sciatic nerve axotomy in the mouse. Exons with upstream promoter regions containing the Sox binding motif 5′-AACAAAG-3′ (I, IV, VII and VIII) were increased at 1d or 3d following axotomy. Exons 1 and IV showed the greatest increase and only exon 1 remained elevated at 3d. Luciferase assays showed that Sox11 could activate the most highly regulated exons, I and IV, and that this activation was reduced by mutation of putative Sox binding sites. Exon expression in injured DRG neurons had some overlap with Neuro2a cells that overexpress Sox11, showing elevation in exon IV and VII transcripts. These findings indicate cell type and contextual specificity of Sox11 in modulation of BDNF transcription. PMID:22331573

  12. Amelogenin Exon4 Forms a Novel miRNA That Directs Ameloblast and Osteoblast Differentiation.

    PubMed

    Le, M H; Warotayanont, R; Stahl, J; Den Besten, P K; Nakano, Y

    2016-04-01

    Amelogenins constitute the major portion of secretory enamel matrix proteins and are known to be highly alternative spliced. Of all the alternatively spliced forms of amelogenins, exon4 is most commonly spliced out. Our analyses of the exon4 sequence led us to hypothesize that when spliced out, exon4 may generate a novel mature miRNA. To explore this possibility, we used in vivo mouse models (wild-type and Amel knockout mice) and in vitro cell culture to investigate the presence and function of a mature miRNA derived from exon4 (miR-exon4). When ameloblast-like cells (LS8) were transfected with an amelogenin minigene to increase amelogenin synthesis, the transfected cells synthesized miR-exon4. Introduction of a mutation in the conserved CNNC sequence required for primary miRNA recognition, downstream of the mature miR-exon4 sequence, resulted in a significantly reduced production of miR-exon4 in the transfected cells. In vivo, miR-exon4 was most highly amplified from wild-type mouse enamel organs at the secretory stage. In Amel knockout mice, an in vivo model for reduced amelogenin synthesis, we found reduced miR-exon4, with no changes in expression of enamel matrix-related genes. However, expression of Runx2 and its downstream genes Odam and Amtn were significantly downregulated. Transfection of miR-exon4 mimic to the LS8 cells also significantly upregulated Runx2. The mature miR-exon4 as well as Runx2 was also present in mouse osteoblasts with no apparent change in expression level between wild-type and Amel knockout mice. However, transfecting miR-exon4 inhibitor to the MC3T3-E1 osteoblastic cells resulted in a significant downregulation of Runx2 expression. These data indicate that when exon4 is spliced out, as occurs most of the time during alternative splicing of amelogenin pre-mRNA, a novel mature miRNA is generated from exon4. This miR-exon4 may contribute to the differentiation of ameloblasts and osteoblasts through regulation of Runx2 expression. PMID

  13. Exon recognition and nucleocytoplasmic partitioning determine AMPD1 alternative transcript production.

    PubMed Central

    Mineo, I; Holmes, E W

    1991-01-01

    Two mature transcripts are produced from the rat AMP deaminase 1 (AMPD1) gene, one that retains exon 2 and one from which exon 2 has been removed. The ratio of these two transcripts is controlled by stage-specific and tissue-specific signals (I. Mineo, P. R. H. Clarke, R. L. Sabina, and E. W. Holmes, Mol. Cell. Biol. 10:5271-5278, 1990; R. L. Sabina, N. Ogasawara, and E. W. Holmes, Mol. Cell. Biol. 9:2244-2246, 1989). By using transfection studies with native, mutant, and chimeric minigene constructs, two steps in RNA processing that determine the ratio of these two transcripts have been identified. The first step is recognition of this exon in the primary transcript. The primary transcript is subject to alternative splicing in which exon 2 is either recognized and thereby included in the mature mRNA or is ignored and retained in a composite intron containing intron 1-exon 2-intron 2. The following properties of the primary transcript influence exon recognition. (i) Exon 2 is intrinsically difficult to recognize, possibly because of its small size (only 12 bases) and/or a suboptimal 5' donor site at the exon 2-intron 2 boundary. (ii) Intron 2 plays a permissive role in recognition of exon 2 because it is removed at a relatively slow rate, presumably because of the suboptimal polypyrimidine tract in the putative 3' branch site. The second step in RNA processing that influences the ratio of mature transcripts produced from the AMPD1 gene occurs subsequent to the ligation of exon 2 to exon 1. An RNA intermediate, composed of exon 1-exon 2-intron 2-exon 3, is produced in the first processing step, but it is variably retained in the nucleus. Retention of this intermediate in the nucleus is associated with accumulation of the mature mRNA containing exon 2, while cytoplasmic escape of this intermediate is reactions, exon recognition and nucleocytoplasmic partitioning, determine the relative abundance of alternative mRNAs derived from the AMPD1 gene. Images PMID:1922051

  14. Testing for natural selection in human exonic splicing regulators associated with evolutionary rate shifts.

    PubMed

    Ramalho, Rodrigo F; Gelfman, Sahar; de Souza, Jorge E; Ast, Gil; de Souza, Sandro J; Meyer, Diogo

    2013-04-01

    Despite evidence that at the interspecific scale, exonic splicing silencers (ESSs) are under negative selection in constitutive exons, little is known about the effects of slightly deleterious polymorphisms on these splicing regulators. Through the application of a modified version of the McDonald-Kreitman test, we compared the normalized proportions of human polymorphisms and human/rhesus substitutions affecting exonic splicing regulators (ESRs) on sequences of constitutive and alternative exons. Our results show a depletion of substitutions and an enrichment of SNPs associated with ESS gain in constitutive exons. Moreover, we show that this evolutionary pattern is also present in a set of ESRs previously involved in the transition from constitutive to skipped exons in the mammalian lineage. The similarity between these two sets of ESRs suggests that the transition from constitutive to skipped exons in mammals is more frequently associated with the inhibition than with the promotion of splicing signals. This is in accordance with the hypothesis of a constitutive origin of exon skipping and corroborates previous findings about the antagonistic role of certain exonic splicing enhancers. PMID:23529588

  15. Comprehensive exon array data processing method for quantitative analysis of alternative spliced variants

    PubMed Central

    Chen, Ping; Lepikhova, Tatiana; Hu, Yizhou; Monni, Outi; Hautaniemi, Sampsa

    2011-01-01

    Alternative splicing of pre-mRNA generates protein diversity. Dysfunction of splicing machinery and expression of specific transcripts has been linked to cancer progression and drug response. Exon microarray technology enables genome-wide quantification of expression levels of the majority of exons and facilitates the discovery of alternative splicing events. Analysis of exon array data is more challenging than the analysis of gene expression data and there is a need for reliable quantification of exons and alternatively spliced variants. We introduce a novel, computationally efficient methodology, Multiple Exon Array Preprocessing (MEAP), for exon array data pre-processing, analysis and visualization. We compared MEAP with existing pre-processing methods, and validation of six exons and two alternatively spliced variants with qPCR corroborated MEAP expression estimates. Analysis of exon array data from head and neck squamous cell carcinoma (HNSCC) cell lines revealed several transcripts associated with 11q13 amplification, which is related with decreased survival and metastasis in HNSCC patients. Our results demonstrate that MEAP produces reliable expression values at exon, alternatively spliced variant and gene levels, which allows generating novel experimentally testable predictions. PMID:21745820

  16. A Relaxed Active Site After Exon Ligation by the Group I Intron

    SciTech Connect

    Lipchock,S.; Strobel, S.

    2008-01-01

    During RNA maturation, the group I intron promotes two sequential phosphorotransfer reactions resulting in exon ligation and intron release. Here, we report the crystal structure of the intron in complex with spliced exons and two additional structures that examine the role of active-site metal ions during the second step of RNA splicing. These structures reveal a relaxed active site, in which direct metal coordination by the exons is lost after ligation, while other tertiary interactions are retained between the exon and the intron. Consistent with these structural observations, kinetic and thermodynamic measurements show that the scissile phosphate makes direct contact with metals in the ground state before exon ligation and in the transition state, but not after exon ligation. Despite no direct exonic interactions and even in the absence of the scissile phosphate, two metal ions remain bound within the active site. Together, these data suggest that release of the ligated exons from the intron is preceded by a change in substrate-metal coordination before tertiary hydrogen bonding contacts to the exons are broken.

  17. Exon dosage analysis of parkin gene in Chinese sporadic Parkinson's disease.

    PubMed

    Guo, Ji-Feng; Dong, Xiao-Li; Xu, Qian; Li, Nan; Yan, Xin-Xiang; Xia, Kun; Tang, Bei-Sha

    2015-09-14

    Parkin gene mutations are by far the most common mutations in both familial Parkinson's disease (PD) and sporadic PD. Approximately, 50% of parkin mutations is exon dosage mutations (i.e., deletions and duplications of entire exons). Here, we first established a MLPA assay for quick detection of parkin exon rearrangements. Then, we studied parkin exon dosage mutations in 755 Chinese sporadic PDdisease patients using the established MLPA assay. We found that there were 25 (3.3%) patients with exon dosage alterations including deletions and duplications, 20 (11.4%) patients with exon rearrangements in 178 early-onset patients, and 5 (0.86%) patients with exon rearrangement mutations in 579 later-onset patients. The percentage of individuals with parkin dosage mutations is more than 33% when the age at onset is less than 30 years old, but less than 7% when the age at onset is more than 30. In these mutations, deletion is the main mutational style, especially in exon 2-5. Our results indicated that exon dosage mutations in parkin gene might be the main cause for sporadic PD, especially in EOP. PMID:26240990

  18. Coding exon-structure aware realigner (CESAR) utilizes genome alignments for accurate comparative gene annotation

    PubMed Central

    Sharma, Virag; Elghafari, Anas; Hiller, Michael

    2016-01-01

    Identifying coding genes is an essential step in genome annotation. Here, we utilize existing whole genome alignments to detect conserved coding exons and then map gene annotations from one genome to many aligned genomes. We show that genome alignments contain thousands of spurious frameshifts and splice site mutations in exons that are truly conserved. To overcome these limitations, we have developed CESAR (Coding Exon-Structure Aware Realigner) that realigns coding exons, while considering reading frame and splice sites of each exon. CESAR effectively avoids spurious frameshifts in conserved genes and detects 91% of shifted splice sites. This results in the identification of thousands of additional conserved exons and 99% of the exons that lack inactivating mutations match real exons. Finally, to demonstrate the potential of using CESAR for comparative gene annotation, we applied it to 188 788 exons of 19 865 human genes to annotate human genes in 99 other vertebrates. These comparative gene annotations are available as a resource (http://bds.mpi-cbg.de/hillerlab/CESAR/). CESAR (https://github.com/hillerlab/CESAR/) can readily be applied to other alignments to accurately annotate coding genes in many other vertebrate and invertebrate genomes. PMID:27016733

  19. Coding exon-structure aware realigner (CESAR) utilizes genome alignments for accurate comparative gene annotation.

    PubMed

    Sharma, Virag; Elghafari, Anas; Hiller, Michael

    2016-06-20

    Identifying coding genes is an essential step in genome annotation. Here, we utilize existing whole genome alignments to detect conserved coding exons and then map gene annotations from one genome to many aligned genomes. We show that genome alignments contain thousands of spurious frameshifts and splice site mutations in exons that are truly conserved. To overcome these limitations, we have developed CESAR (Coding Exon-Structure Aware Realigner) that realigns coding exons, while considering reading frame and splice sites of each exon. CESAR effectively avoids spurious frameshifts in conserved genes and detects 91% of shifted splice sites. This results in the identification of thousands of additional conserved exons and 99% of the exons that lack inactivating mutations match real exons. Finally, to demonstrate the potential of using CESAR for comparative gene annotation, we applied it to 188 788 exons of 19 865 human genes to annotate human genes in 99 other vertebrates. These comparative gene annotations are available as a resource (http://bds.mpi-cbg.de/hillerlab/CESAR/). CESAR (https://github.com/hillerlab/CESAR/) can readily be applied to other alignments to accurately annotate coding genes in many other vertebrate and invertebrate genomes. PMID:27016733

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

    PubMed

    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. PMID:24858907

  1. Exon capture optimization in amphibians with large genomes.

    PubMed

    McCartney-Melstad, Evan; Mount, Genevieve G; Shaffer, H Bradley

    2016-09-01

    Gathering genomic-scale data efficiently is challenging for nonmodel species with large, complex genomes. Transcriptome sequencing is accessible for organisms with large genomes, and sequence capture probes can be designed from such mRNA sequences to enrich and sequence exonic regions. Maximizing enrichment efficiency is important to reduce sequencing costs, but relatively few data exist for exon capture experiments in nonmodel organisms with large genomes. Here, we conducted a replicated factorial experiment to explore the effects of several modifications to standard protocols that might increase sequence capture efficiency for amphibians and other taxa with large, complex genomes. Increasing the amounts of c0 t-1 repetitive sequence blocker and individual input DNA used in target enrichment reactions reduced the rates of PCR duplication. This reduction led to an increase in the percentage of unique reads mapping to target sequences, essentially doubling overall efficiency of the target capture from 10.4% to nearly 19.9% and rendering target capture experiments more efficient and affordable. Our results indicate that target capture protocols can be modified to efficiently screen vertebrates with large genomes, including amphibians. PMID:27223337

  2. JuncDB: an exon–exon junction database

    PubMed Central

    Chorev, Michal; Guy, Lotem; Carmel, Liran

    2016-01-01

    Intron positions upon the mRNA transcript are sometimes remarkably conserved even across distantly related eukaryotic species. This has made the comparison of intron–exon architectures across orthologous transcripts a very useful tool for studying various evolutionary processes. Moreover, the wide range of functions associated with introns may confer biological meaning to evolutionary changes in gene architectures. Yet, there is currently no database that offers such comparative information. Here, we present JuncDB (http://juncdb.carmelab.huji.ac.il/), an exon–exon junction database dedicated to the comparison of architectures between orthologous transcripts. It covers nearly 40 000 sets of orthologous transcripts spanning 88 eukaryotic species. JuncDB offers a user-friendly interface, access to detailed information, instructive graphical displays of the comparative data and easy ways to download data to a local computer. In addition, JuncDB allows the analysis to be carried out either on specific genes, or at a genome-wide level for any selected group of species. PMID:26519469

  3. EXONSAMPLER: a computer program for genome-wide and candidate gene exon sampling for targeted next-generation sequencing.

    PubMed

    Cosart, Ted; Beja-Pereira, Albano; Luikart, Gordon

    2014-11-01

    The computer program EXONSAMPLER automates the sampling of thousands of exon sequences from publicly available reference genome sequences and gene annotation databases. It was designed to provide exon sequences for the efficient, next-generation gene sequencing method called exon capture. The exon sequences can be sampled by a list of gene name abbreviations (e.g. IFNG, TLR1), or by sampling exons from genes spaced evenly across chromosomes. It provides a list of genomic coordinates (a bed file), as well as a set of sequences in fasta format. User-adjustable parameters for collecting exon sequences include a minimum and maximum acceptable exon length, maximum number of exonic base pairs (bp) to sample per gene, and maximum total bp for the entire collection. It allows for partial sampling of very large exons. It can preferentially sample upstream (5 prime) exons, downstream (3 prime) exons, both external exons, or all internal exons. It is written in the Python programming language using its free libraries. We describe the use of EXONSAMPLER to collect exon sequences from the domestic cow (Bos taurus) genome for the design of an exon-capture microarray to sequence exons from related species, including the zebu cow and wild bison. We collected ~10% of the exome (~3 million bp), including 155 candidate genes, and ~16,000 exons evenly spaced genomewide. We prioritized the collection of 5 prime exons to facilitate discovery and genotyping of SNPs near upstream gene regulatory DNA sequences, which control gene expression and are often under natural selection. PMID:24751285

  4. WNT10A exonic variant increases the risk of keratoconus by decreasing corneal thickness.

    PubMed

    Cuellar-Partida, Gabriel; Springelkamp, Henriët; Lucas, Sionne E M; Yazar, Seyhan; Hewitt, Alex W; Iglesias, Adriana I; Montgomery, Grant W; Martin, Nicholas G; Pennell, Craig E; van Leeuwen, Elisabeth M; Verhoeven, Virginie J M; Hofman, Albert; Uitterlinden, André G; Ramdas, Wishal D; Wolfs, Roger C W; Vingerling, Johannes R; Brown, Matthew A; Mills, Richard A; Craig, Jamie E; Klaver, Caroline C W; van Duijn, Cornelia M; Burdon, Kathryn P; MacGregor, Stuart; Mackey, David A

    2015-09-01

    Keratoconus is a degenerative eye condition which results from thinning of the cornea and causes vision distortion. Treatments such as ultraviolet (UV) cross-linking have proved effective for management of keratoconus when performed in early stages of the disease. The central corneal thickness (CCT) is a highly heritable endophenotype of keratoconus, and it is estimated that up to 95% of its phenotypic variance is due to genetics. Genome-wide association efforts of CCT have identified common variants (i.e. minor allele frequency (MAF) >5%). However, these studies typically ignore the large set of exonic variants whose MAF is usually low. In this study, we performed a CCT exome-wide association analysis in a sample of 1029 individuals from a population-based study in Western Australia. We identified a genome-wide significant exonic variant rs121908120 (P = 6.63 × 10(-10)) in WNT10A. This gene is 437 kb from a gene previously associated with CCT (USP37). We showed in a conditional analysis that the WNT10A variant completely accounts for the signal previously seen at USP37. We replicated our finding in independent samples from the Brisbane Adolescent Twin Study, Twin Eye Study in Tasmania and the Rotterdam Study. Further, we genotyped rs121908120 in 621 keratoconus cases and compared the frequency to a sample of 1680 unscreened controls from the Queensland Twin Registry. We found that rs121908120 increases the risk of keratoconus two times (odds ratio 2.03, P = 5.41 × 10(-5)). PMID:26049155

  5. Requirements for mini-exon inclusion in potato invertase mRNAs provides evidence for exon-scanning interactions in plants.

    PubMed Central

    Simpson, C G; Hedley, P E; Watters, J A; Clark, G P; McQuade, C; Machray, G C; Brown, J W

    2000-01-01

    Invertases are responsible for the breakdown of sucrose to fructose and glucose. In all but one plant invertase gene, the second exon is only 9 nt in length and encodes three amino acids of a five-amino-acid sequence that is highly conserved in all invertases of plant origin. Sequences responsible for normal splicing (inclusion) of exon 2 have been investigated in vivo using the potato invertase, invGF gene. The upstream intron 1 is required for inclusion whereas the downstream intron 2 is not. Mutations within intron 1 have identified two sequence elements that are needed for inclusion: a putative branchpoint sequence and an adjacent U-rich region. Both are recognized plant intron splicing signals. The branchpoint sequence lies further upstream from the 3' splice site of intron 1 than is normally seen in plant introns. All dicotyledonous plant invertase genes contain this arrangement of sequence elements: a distal branchpoint sequence and adjacent, downstream U-rich region. Intron 1 sequences upstream of the branchpoint and sequences in exons 1, 2, or 3 do not determine inclusion, suggesting that intron or exon splicing enhancer elements seen in vertebrate mini-exon systems are absent. In addition, mutation of the 3' and 5' splice sites flanking the mini-exon cause skipping of the mini-exon, suggesting that both splice sites are required. The branchpoint/U-rich sequence is able to promote splicing of mini-exons of 6, 3, and 1 nt in length and of a chicken cTNT mini-exon of 6 nt. These sequence elements therefore act as a splicing enhancer and appear to function via interactions between factors bound at the branchpoint/U-rich region and at the 5' splice site of intron 2, activating removal of this intron followed by removal of intron 1. This first example of splicing of a plant mini-exon to be analyzed demonstrates that particular arrangement of standard plant intron splicing signals can drive constitutive splicing of a mini-exon. PMID:10744026

  6. Regulation of Fibronectin EDA Exon Alternative Splicing: Possible Role of RNA Secondary Structure for Enhancer Display

    PubMed Central

    Muro, Andrés F.; Caputi, Massimo; Pariyarath, Rajalakshmi; Pagani, Franco; Buratti, Emanuele; Baralle, Francisco E.

    1999-01-01

    The fibronectin primary transcript undergoes alternative splicing in three noncoordinated sites: the cassette-type EDA and EDB exons and the more complex IIICS region. We have shown previously that an 81-nucleotide region within the EDA exon is necessary for exon recognition and that this region contains at least two splicing-regulatory elements: a polypurinic enhancer (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). Here, we have analyzed the function of both elements in different cell types. We have mapped the ESS to the nucleotide level, showing that a single base change is sufficient to abolish its function. Testing of the ESE and ESS elements in heterologous exons, individually or as part of the complete EDA regulatory region, showed that only the ESE element is active in different contexts. Functional studies coupled to secondary-structure enzymatic analysis of the EDA exon sequence variants suggest that the role of the ESS element may be exclusively to ensure the proper RNA conformation and raise the possibility that the display of the ESE element in a loop position may represent a significant feature of the exon splicing-regulatory region. PMID:10082532

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

    PubMed Central

    BARZEGAR, Mohammad; HABIBI, Parinaz; BONYADY, Mortaza; TOPCHIZADEH, Vahideh; SHIVA, Shadi

    2015-01-01

    Objective Duchene 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 &Methods In 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. Results Deletions 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 single-exon 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. Conclusion This 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. PMID:25767538

  8. Isolation of genes from the Batten candidate region using exon amplification

    SciTech Connect

    Lerner, T.J.; D`Arigo, K.L.; Haines, J.L.

    1995-06-05

    In order to identify genes originating from the Batten disease candidate region, we have used the technique of exon amplification to identify transcribed sequences. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. The source of DNA for these experiments was a collection of chromosome 16 cosmid contigs isolated by the direct subcloning of region-specific yeast artificial chromosomes (YACs) and hybridization of inter-alu PCR products from these YACs to the flow-sorted Los Alamos chromosome 16 cosmid library. We are now using the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes. 23 refs., 1 fig.

  9. Characterization of the exon structure of the Menkes disease gene using vectorette PCR

    SciTech Connect

    Tuemer, Z.; Tonnesen, T.; Horn, N.

    1995-04-10

    The gene defective in Menkes disease, an X-linked recessive disturbance of copper metabolism, has been isolated and predicted to encode a copper-binding P-type ATPase. We determined the complete exon-intron structure of the Menkes disease gene, which spans about 150 kb of genomic DNA. The gene contains 23 exons, and the ATG start codon is in the second exon. All of the exon-intron boundaries were sequenced and conformed to the GT/AT rule, except for the 5{prime} splice site of intron 9. A preliminary comparison demonstrated a striking similarity between the exon structures of the Menkes and Wilson disease genes, giving insight into their evolution. 33 refs., 3 figs., 2 tabs.

  10. CoNVaDING: Single Exon Variation Detection in Targeted NGS Data.

    PubMed

    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. PMID:26864275

  11. Finding intron/exon splice junctions using INFO, INterruption Finder and Organizer.

    PubMed

    Laub, M T; Smith, D W

    1998-01-01

    INFO, INterruption Finder and Organizer, has been used to find coding sequence intron-exon splice junctions in human and other DNA by comparing the six conceptual translations of the input DNA sequence with sequences in protein databanks using a similarity matrix and windowing algorithm. Similarities detected both delineate position of the gene and provide clues as to the function of the gene product. In addition to use of a standard similarity matrix and windowing algorithm, INFO uses two novel steps, the MiniLibrary and Reverse Sequence steps, to enhance identification of small exons and to improve precision of junction nucleotide delineation. Exons as small as about 30 bases can be reliably found, and > 90% of junctions are precisely identified when canonical splice junction information is used. With the MiniLibrary and Reverse Sequence steps, INFO parameters need not be optimized by the user. In comparative test runs using 19 human DNA sequences, INFO found 108 of 111 exons, with 0 reported false positives, compared with 111 exons and 51 false positives for BLASTX, 99 exons and 6 false positives for GRAIL II, 77 exons and 24 false positives for GeneMark, 61 exons and 9 false positives for GeneID, and 105 exons and 6 false positives for PROCRUSTES. The correlation coefficient for finding and positioning these 111 exons was greater than 98% for INFO. Comparable results were obtained in test runs of 13 nonhuman DNA sequences. INFO is applicable to DNA from any species, will become more robust as sequence databanks expand, and complements other heuristic approaches. PMID:9672834

  12. The exon-intron organization of the human erythroid [beta]-spectrin gene

    SciTech Connect

    Amin, K.M.; Forget, B.G. ); Scarpa, A.L.; Curtis, P.J. ); Winkelmann, J.C. )

    1993-10-01

    The human erythrocyte [beta]-spectrin gene DNA has been cloned from overlapping human genomic phage and cosmid recombinants. The entire erythroid [beta]-spectrin mRNA is encoded by 32 exons that range in size from 49 to 871 bases. The exon/intron junctions have been identified and the exons mapped. There is no correlation between intron positions and the repeat units of 106 amino acids within domain II of the [beta]-spectrin gene. The scatter of the introns over the 17 repeats argues against the 106-amino-acid unit representing a minigene that underwent repeated duplication resulting in the present [beta]-spectrin gene. In fact, the two largest exons, exon 14 (871 bp) and 16 (757 bp), extend over 4 and 3 repeat units of 106 amino acids, respectively, while repeat [beta]10 is encoded by 4 exons. No single position of an intron in the [beta]-spectrin gene is conserved between any of the 17 [beta]-spectrin and 22 [alpha]-spectrin repeat units. The nucleotide sequences of the exon/intron boundaries conform to the consensus splice site sequences except for exon 20, whose 5[prime] donor splice-site sequence begins with GC. The [beta]-spectrin isoform present in the human brain, the skeletal muscle, and the cardiac muscle is an alternatively spliced product of the erythroid [beta]-spectrin gene. This splice site is located within the coding sequences of exon 32 and its utilization in nonerythroid tissues leads to the use of 4 additional downstream exons with a size range of 44 to 530 bp. 55 refs., 3 figs., 3 tabs.

  13. The Contribution of Exon-Skipping Events on Chromosome 22 to Protein Coding Diversity

    PubMed Central

    Hide, Winston A.; Babenko, Vladimir N.; van Heusden, Peter A.; Seoighe, Cathal; Kelso, Janet F.

    2001-01-01

    Completion of the human genome sequence provides evidence for a gene count with lower bound 30,000–40,000. Significant protein complexity may derive in part from multiple transcript isoforms. Recent EST based studies have revealed that alternate transcription, including alternative splicing, polyadenylation and transcription start sites, occurs within at least 30–40% of human genes. Transcript form surveys have yet to integrate the genomic context, expression, frequency, and contribution to protein diversity of isoform variation. We determine here the degree to which protein coding diversity may be influenced by alternate expression of transcripts by exhaustive manual confirmation of genome sequence annotation, and comparison to available transcript data to accurately associate skipped exon isoforms with genomic sequence. Relative expression levels of transcripts are estimated from EST database representation. The rigorous in silico method accurately identifies exon skipping using verified genome sequence. 545 genes have been studied in this first hand-curated assessment of exon skipping on chromosome 22. Combining manual assessment with software screening of exon boundaries provides a highly accurate and internally consistent indication of skipping frequency. 57 of 62 exon skipping events occur in the protein coding regions of 52 genes. A single gene, (FBXO7) expresses an exon repetition. 59% of highly represented multi-exon genes are likely to express exon-skipped isoforms in ratios that vary from 1:1 to 1:>100. The proportion of all transcripts corresponding to multi-exon genes that exhibit an exon skip is estimated to be 5%. PMID:11691849

  14. SF2 and SRp55 regulation of CD45 exon 4 skipping during T cell activation.

    PubMed

    Lemaire, R; Winne, A; Sarkissian, M; Lafyatis, R

    1999-03-01

    CD45 is an alternatively spliced membrane phosphatase required for T cell activation. Exons 4, 5 and 6 can be included or skipped from spliced mRNA resulting in several protein isoforms that include or exclude epitopes referred to as RA, RB or RC, respectively. T cells reciprocally express CD45RA or CD45RO (lacking all three exons), corresponding to naive versus memory T cells. Overexpression of the alternative splicing regulators, SF2 or SWAP, induces skipping of CD45 exon 4 in transfected COS cells. We show here that the arginine/serine-rich domain of SWAP and the RNA recognition motifs of SF2 are required for skipping of CD45 exon 4. Unlike SWAP, SF2 specifically regulated alternative splicing of CD45 exon 4, having no effect on a non-regulated exon of CD45 (exon 9). Like SF2 and SWAP, the SR proteins SC35, SRp40 and SRp75, but not SRp55 also induced CD45 exon 4 skipping. In contrast, antisense inhibition of SRp55 induced exon 4 skipping. SF2 and SRp55 proteins were not detectable or expressed at a very low level in freshly isolated CD45RA+ and CD45RO+ T cells. Activation of CD45RA+ T cells shifted CD45 expression from CD45RA to CD45RO, and induced a large increase in expression of both SF2 and SRp55. Thus, SF2 at least in part determines splicing of CD45 exon 4 during T cell activation. SRp55, SR protein phosphorylation, or other splicing factors likely regulate CD45 splicing in CD45RO+ memory T cells. The different SR proteins expressed by memory and activated T cells emphasize the different phenotypes of these cell types that both express CD45RO. PMID:10092085

  15. Contrasting chromatin organization of CpG islands and exons in the human genome

    PubMed Central

    2010-01-01

    Background CpG islands and nucleosome-free regions are both found in promoters. However, their association has never been studied. On the other hand, DNA methylation is absent in promoters but is enriched in gene bodies. Intragenic nucleosomes and their modifications have been recently associated with RNA splicing. Because the function of intragenic DNA methylation remains unclear, I explored the possibility of its involvement in splicing regulation. Results Here I show that CpG islands were associated not only with methylation-free promoters but also with nucleosome-free promoters. Nucleosome-free regions were observed only in promoters containing a CpG island. However, the DNA sequences of CpG islands predicted the opposite pattern, implying a limitation of sequence programs for the determination of nucleosome occupancy. In contrast to the methylation-and nucleosome-free states of CpG-island promoters, exons were densely methylated at CpGs and packaged into nucleosomes. Exon-enrichment of DNA methylation was specifically found in spliced exons and in exons with weak splice sites. The enrichment patterns were less pronounced in initial exons and in non-coding exons, potentially reflecting a lower need for their splicing. I also found that nucleosomes, DNA methylation, and H3K36me3 marked the exons of transcripts with low, medium, and high gene expression levels, respectively. Conclusions Human promoters containing a CpG island tend to remain nucleosome-free as well as methylation-free. In contrast, exons demonstrate a high degree of methylation and nucleosome occupancy. Exonic DNA methylation seems to function together with exonic nucleosomes and H3K36me3 for the proper splicing of transcripts with different expression levels. PMID:20602769

  16. Microsomal epoxide hydrolase (EPHX1), slow (exon 3, 113His) and fast (exon 4, 139Arg) alleles confer susceptibility to squamous cell esophageal cancer

    SciTech Connect

    Jain, Meenu; Tilak, Anup Raj; Upadhyay, Rohit; Kumar, Ashwani; Mittal, Balraj

    2008-07-15

    Genetic polymorphisms in xenobiotic metabolizing enzymes may alter risk of various cancers. Present case-control study evaluated the influence of EPHX1 genetic variations on squamous cell esophageal cancer (ESCC) susceptibility in 107 patients and 320 controls. EPHX1 polymorphic alleles were genotyped by direct sequencing (exon 3, Tyr113His) or PCR-RFLP (exon 4, His139Arg). Patients with exon 3 genotypes (Tyr113His, His113His) and 113His allele were at risk of ESCC (OR{sub Tyr113His} 2.0, 95% CI = 1.2-3.4, p = 0.007; OR{sub His113His} 2.3 95% CI = 1.0-5.2, p = 0.03 and OR{sub His} 1.5, 95% CI = 1.0-2.1, p = 0.01). In contrast, individuals with exon 4, 139Arg allele were at low risk of cancer (OR 0.34, 95% CI = 0.20-0.56, p = 0.001). However, none of haplotype combinations of exon 3 (Tyr113His) and exon 4 (His139Arg) polymorphisms showed modulation of risk for ESCC. Sub-grouping of patients based on anatomical location of tumor predicted that patients with exon 3, His113His and Tyr113His genotypes were at higher risk for developing ESCC tumor at upper and middle third locations (OR 4.4, 95% CI = 1.0-18.5, p = 0.04; OR 2.5, 95% CI = 1.3-5.0, p = 0.005 respectively). The frequency of exon 4, His139Arg genotype was significantly lower in ESCC patients with lower third tumor location as compared to controls (14.8% vs. 36.3%, p = 0.02). In case-only study, gene-environment interaction of EPHX1 genotypes with tobacco, alcohol and occupational exposures did not appear to modulate the cancer susceptibility. In conclusion, exon 3, Tyr113His genotype was associated with higher risk of ESCC particularly at upper and middle-third anatomical locations of tumor. However, His139Arg genotype of exon 4, exhibited low risk for ESCC as well as its clinical characteristics.

  17. Development of a novel PTT assay for mutation detection in PALB2 large exons and PALB2 screening in medullary breast cancer.

    PubMed

    Poumpouridou, Nikoleta; Goutas, Nikolaos; Tsionou, Christina; Dimas, Kleanthi; Lianidou, Evi; Kroupis, Christos

    2016-04-01

    Beyond BRCA1 and BRCA2 genes, PALB2 (Partner and localizer of BRCA2) emerges as the third breast cancer susceptibility gene due to its role in the same DNA repair pathway: homologous recombination. In most populations studied so far, PALB2 mutations are detected in 1-2% of BRCA negative female patients. PALB2 gene contains 13 exons; exons 4 and 5 consist 65% of the coding area. We developed a protein truncation test (PTT) for quick screening of truncating pathogenic mutations of these two large exons. Specific primers were de novo, in silico designed and the PTT-PCR products were translated in the presence of biotinylated lysine and detected colorimetrically. The assay was initially tested in 30 patients with hereditary breast cancer, negative for BRCA mutations and then, in 17 patients with the rare medullary breast cancer subtype. Small PALB2 exons were screened with high-resolution melting curve analysis (HRMA) and the large DNA rearrangements with multiplex ligation-dependent probe amplification (MLPA). Any alterations detected were verified by Sanger DNA Sequencing. The developed PTT methodology is highly specific for clinical significant mutations; positive control samples that produce truncated PALB2 peptides were correctly identified and the method was accurate when compared to DNA sequencing. We did not detect any deleterious PALB2 mutation in both groups of patients. HRMA and MLPA were also negative for all tested samples. However, our novel, fast and cost-effective PTT method for pathogenic mutation detection of the two large PALB2 exons can be applied in screening of a large number of breast cancer patients. PMID:26573693

  18. The Evolutionary Fate of Alternatively Spliced Homologous Exons after Gene Duplication

    PubMed Central

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

    2015-01-01

    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. PMID:25931610

  19. 11p15-subband specific search for transcribed sequences using exon trapping

    SciTech Connect

    Loebbert, R.; Prawitt, D.; Monroe, D.

    1994-09-01

    Evidence from cytogenetic and molecular data suggest that the region 11p15 contains genes involved in different disorders, like Beckwith-Wiedemann syndrome (BWS), long QT syndrome (LQT), Usher syndrome type I and tumor development. Focusing on the subregion 11p15.1, we are isolating and characterizing new transcribed sequences. The applied strategy includes exon amplification and subsequent PCR screening of cDNA libraries. So far 100 YACs and 38 cosmid clones from 11p15.1-15.3 have been collected and are currently arrayed. 16 cosmids have been analyzed for transcribed sequences using the exon amplification scheme developed by Buckler et al. (1991). We were able to identify 18 exons that contain correct open reading frames and map back to the cosmid clones. A data base search revealed that two exons represent parts of known genes from this region (ST5 and AMPD3). Moreover, we identified one exon that represents an EGF-like repeat with homologies to various proteins. Using PCR and primers from the exon sequences, a fetal brain library, which has been arranged in the form of hierarchic arrayed phage pools, was screened. Up to now, two cDNA clones corresponding to different exons were isolated and are currently sequenced.

  20. Oncogene- and drug resistance-associated alternative exon usage in acute myeloid leukemia (AML).

    PubMed

    Mohamed, Aminetou Mint; Balsat, Marie; Thenoz, Morgan; Koering, Catherine; Payen-Gay, Lea; Cheok, Meyling; Mortada, Hussein; Auboeuf, Didier; Pinatel, Christiane; El-Hamri, Mohamed; Dumontet, Charles; Cros, Emeline; Flandrin-Gresta, Pascale; Nibourel, Olivier; Preudhomme, Claude; Michallet, Mauricette; Thomas, Xavier; Nicolini, Franck; Solly, Françoise; Guyotat, Denis; Campos, Lydia; Wattel, Eric; Mortreux, Franck

    2016-01-19

    In addition to spliceosome gene mutations, oncogene expression and drug resistance in AML might influence exon expression. We performed exon-array analysis and exon-specific PCR (ESPCR) to identify specific landscapes of exon expression that are associated with DEK and WT1 oncogene expression and the resistance of AML cells to AraC, doxorubicin or azacitidine. Data were obtained for these five conditions through exon-array analysis of 17 cell lines and 24 patient samples and were extended through qESPCR of samples from 152 additional AML cases. More than 70% of AEUs identified by exon-array were technically validated through ESPCR. In vitro, 1,130 to 5,868 exon events distinguished the 5 conditions from their respective controls while in vivo 6,560 and 9,378 events distinguished chemosensitive and chemoresistant AML, respectively, from normal bone marrow. Whatever the cause of this effect, 30 to 80% of mis-spliced mRNAs involved genes unmodified at the whole transcriptional level. These AEUs unmasked new functional pathways that are distinct from those generated by transcriptional deregulation. These results also identified new putative pathways that could help increase the understanding of the effects mediated by DEK or WT1, which may allow the targeting of these pathways to prevent resistance of AML cells to chemotherapeutic agents. PMID:26284582

  1. Reengineering a transmembrane protein to treat muscular dystrophy using exon skipping.

    PubMed

    Gao, Quan Q; Wyatt, Eugene; Goldstein, Jeff A; LoPresti, Peter; Castillo, Lisa M; Gazda, Alec; Petrossian, Natalie; Earley, Judy U; Hadhazy, Michele; Barefield, David Y; Demonbreun, Alexis R; Bönnemann, Carsten; Wolf, Matthew; McNally, Elizabeth M

    2015-11-01

    Exon skipping uses antisense oligonucleotides as a treatment for genetic diseases. The antisense oligonucleotides used for exon skipping are designed to bypass premature stop codons in the target RNA and restore reading frame disruption. Exon skipping is currently being tested in humans with dystrophin gene mutations who have Duchenne muscular dystrophy. For Duchenne muscular dystrophy, the rationale for exon skipping derived from observations in patients with naturally occurring dystrophin gene mutations that generated internally deleted but partially functional dystrophin proteins. We have now expanded the potential for exon skipping by testing whether an internal, in-frame truncation of a transmembrane protein γ-sarcoglycan is functional. We generated an internally truncated γ-sarcoglycan protein that we have termed Mini-Gamma by deleting a large portion of the extracellular domain. Mini-Gamma provided functional and pathological benefits to correct the loss of γ-sarcoglycan in a Drosophila model, in heterologous cell expression studies, and in transgenic mice lacking γ-sarcoglycan. We generated a cellular model of human muscle disease and showed that multiple exon skipping could be induced in RNA that encodes a mutant human γ-sarcoglycan. Since Mini-Gamma represents removal of 4 of the 7 coding exons in γ-sarcoglycan, this approach provides a viable strategy to treat the majority of patients with γ-sarcoglycan gene mutations. PMID:26457733

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

    PubMed

    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. PMID:26365037

  3. Runaway evolution of the male-specific exon of the doublesex gene in Diptera

    PubMed Central

    Hughes, Austin L.

    2010-01-01

    In Diptera (Insecta), alternatively spliced male-specific and female-specific products of the doublesex (dsx) gene play key role in regulating development of the adult genital structures from the genital disc. Analysis of the pattern of nucleotide substitution of different domains of the dsx gene in 29 dipteran species showed that, over short evolutionary times, purifying selection predominated on the domain common to both sexes, the female-specific exons, and the and male-specific exon. However, over longer the evolutionary time frames represented by between-family comparisons, the male-specific exon accumulated nonsynonymous substitutions at a much more rapid rate than either the common domain or the female-specific exon. Overall, the accumulation of nonsynonymous substitutions in the male-specific exon occurred at a significantly greater than linear rate relative to the common domain, whereas the accumulation of nonsynonymous substitutions in the female-specific exon occurred at less than linear rate relative to the common domain. The evolution of the male-specific exon of dsx thus shows a pattern reminiscent of that seen in the “runaway” evolution of male secondary sexual characters at the morphological level, consistent with the hypothesis that female choice is an important factor in the morphological diversification of insect male genitalia. PMID:21059384

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

    PubMed

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

    2015-06-01

    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. PMID:25931610

  5. Dystrophin-deficient large animal models: translational research and exon skipping

    PubMed Central

    Yu, Xinran; Bao, Bo; Echigoya, Yusuke; Yokota, Toshifumi

    2015-01-01

    Duchenne muscular dystrophy (DMD) is an X-linked recessive genetic disorder caused by mutations in the dystrophin gene. Affecting approximately 1 in 3,600-9337 boys, DMD patients exhibit progressive muscle degeneration leading to fatality as a result of heart or respiratory failure. Despite the severity and prevalence of the disease, there is no cure available. While murine models have been successfully used in illustrating the mechanisms of DMD, their utility in DMD research is limited due to their mild disease phenotypes such as lack of severe skeletal muscle and cardiac symptoms. To address the discrepancy between the severity of disease displayed by murine models and human DMD patients, dystrophin-deficient dog models with a splice site mutation in intron 6 were established. Examples of these are Golden Retriever muscular dystrophy and beagle-based Canine X-linked muscular dystrophy. These large animal models are widely employed in therapeutic DMD research due to their close resemblance to the severity of human patient symptoms. Recently, genetically tailored porcine models of DMD with deleted exon 52 were developed by our group and others, and can potentially act as a new large animal model. While therapeutic outcomes derived from these large animal models can be more reliably extrapolated to DMD patients, a comprehensive understanding of these models is still needed. This paper will discuss recent progress and future directions of DMD studies with large animal models such as canine and porcine models. PMID:26396664

  6. Structure and functional evaluation of porcine NANOG that is a single-exon gene and has two pseudogenes.

    PubMed

    Yang, Fan; Zhang, Jinglong; Liu, Yajun; Cheng, De; Wang, Huayan

    2015-02-01

    Nanog plays an important role in maintaining the pluripotency of murine and human embryonic stem cells. However, the molecular features and transcriptional regulation of the NANOG gene have not been well investigated in pig. Here, we report, for the first time, that porcine NANOG is encoded by a single exon gene (SEG) mapped on chromosome 1 and has two daughter genes, one pseudogene NANOGP1 on chromosome 5 and one tandem duplicate on chromosome 1. The duplicated pseudogene NANOGP2 has high sequence similarity to NANOG, but does not encode a functional protein due to deletions and in-frame stop codons. The NANOGP1 contains four exons and three introns, but is short of the homeodomain sequence. Transcriptome analysis confirmed that NANOG mRNA in porcine iPS cells is transcribed from the SEG NANOG, but not from NANOGP1, because the NANOGP1 promoter is highly methylated, as confirmed by global DNA methylation analysis. The NANOG protein encoded by NANOG retains N, H, and C1/W/C2 domains. The H domain is required for nuclear translocation, while the C1/W/C2 domain ensures the NANOG regulatory function. Overexpression of NANOG in porcine embryonic fibroblasts promoted upregulation of its target genes SOX2, KLF4, and c-MYC. In conclusion, the functional porcine NANOG that is different in chromosomal structure from mouse and human genes is a single exon gene and encodes the functional NANOG protein that can be specifically regulated by OCT4/SOX2, and can promote the activation of target pluripotent factors in vivo. PMID:25542179

  7. Changes in exon–intron structure during vertebrate evolution affect the splicing pattern of exons

    PubMed Central

    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

  8. Bodywide skipping of exons 45-55 in dystrophic mdx52 mice by systemic antisense delivery.

    PubMed

    Aoki, Yoshitsugu; Yokota, Toshifumi; Nagata, Tetsuya; Nakamura, Akinori; Tanihata, Jun; Saito, Takashi; Duguez, Stephanie M R; Nagaraju, Kanneboyina; Hoffman, Eric P; Partridge, Terence; Takeda, Shin'ichi

    2012-08-21

    Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self- or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model. PMID:22869723

  9. The expression of the human steroid sulfatase-encoding gene is driven by alternative first exons.

    PubMed

    Dalla Valle, Luisa; Toffolo, Vania; Nardi, Alessia; Fiore, Cristina; Armanini, Decio; Belvedere, Paola; Colombo, Lorenzo

    2007-10-01

    We have analyzed steroid sulfatase (STS) gene transcription in 10 human tissues: ovary, adrenal cortex, uterus, thyroid, liver, pancreas, colon, mammary gland, dermal papilla of the hair follicle, and peripheral mononuclear leukocytes. Overall, six different promoters were found to drive STS expression, giving rise to transcripts with unique first exons that were labeled 0a, 0b, 0c, 1a, 1c, and 1d, of which the last two and 0c are newly reported. All of them, except exon 1d, vary in length owing to the occurrence of multiple transcriptional start sites. While placental exon 1a is partially coding, the other five first exons are all untranslated. Three of these (0a, 0b, and 0c) are spliced to the common partially coding exon 1b, whereas the other two (1c and 1d) are spliced to the coding exon 2, which occurs in all transcripts. Whatever the ATG actually used, the differences are restricted to the signal peptide which is post-transcriptionally cleaved. Transcripts with exons 0a and 0b have the broadest tissue distribution, occurring, in 6 out of the 12 tissues so far investigated, while the other first exons are restricted to one or two tissues. The proximal promoter of each first exon was devoid of TATA box or initiator element and lacked consensus elements for transcription factors related to steroidogenesis, suggesting that regulatory sequences are probably placed at greater distance. In conclusion, the regulation of STS transcription appears to be more complex than previously thought, suggesting that this enzyme plays a substantial role in intercellular integration. PMID:17601726

  10. DNA Aptamers against Exon v10 of CD44 Inhibit Breast Cancer Cell Migration

    PubMed Central

    Iida, Joji; Clancy, Rebecca; Dorchak, Jesse; Somiari, Richard I.; Somiari, Stella; Cutler, Mary Lou; Mural, Richard J.; Shriver, Craig D.

    2014-01-01

    CD44 adhesion molecules are expressed in many breast cancer cells and have been demonstrated to play a key role in regulating malignant phenotypes such as growth, migration, and invasion. CD44 is an integral transmembrane protein encoded by a single 20-exon gene. The diversity of the biological functions of CD44 is the result of the various splicing variants of these exons. Previous studies suggest that exon v10 of CD44 plays a key role in promoting cancer invasion and metastasis, however, the molecular mechanisms are not clear. Given the fact that exon v10 is in the ectodomain of CD44, we hypothesized that CD44 forms a molecular complex with other cell surface molecules through exon v10 in order to promote migration of breast cancer cells. In order to test this hypothesis, we selected DNA aptamers that specifically bound to CD44 exon v10 using Systematic Evolution of Ligands by Exponential Enrichment (SELEX). We selected aptamers that inhibited migration of breast cancer cells. Co-immunoprecipitation studies demonstrated that EphA2 was co-precipitated with CD44. Pull-down studies demonstrated that recombinant CD44 exon v10 bound to EphA2 and more importantly aptamers that inhibited migration also prevented the binding of EphA2 to exon v10. These results suggest that CD44 forms a molecular complex with EphA2 on the breast cancer cell surface and this complex plays a key role in enhancing breast cancer migration. These results provide insight not only for characterizing mechanisms of breast cancer migration but also for developing target-specific therapy for breast cancers and possibly other cancer types expressing CD44 exon v10. PMID:24586375

  11. Exon skipping through the creation of a putative exonic splicing silencer as a consequence of the cystic fibrosis mutation R553X.

    PubMed

    Aznarez, Isabel; Zielenski, Julian; Rommens, Johanna M; Blencowe, Benjamin J; Tsui, Lap-Chee

    2007-05-01

    Nonsense mutations that occur more than 50 bases upstream of terminal spliced junctions are generally thought to lead to degradation of the corresponding transcripts by the process of nonsense-mediated mRNA decay. It has also been proposed that some nonsense mutations may affect splicing by the process of nonsense-associated altered splicing (NAS), or by the disruption of a splicing regulatory element. In this study, the effect of the R553X mutation on the splicing of exon 11 of the cystic fibrosis transmembrane conductance regulator gene was investigated. Evidence that R553X causes exon 11 to skip through the creation of a putative exonic splicing silencer (ESS) was provided. The putative ESS appears to be active when located immediately upstream of a 5' splice site. These findings argue against the possibility that R553X-associated exon 11 skipping is caused by NAS. The study further suggests that aminoglycoside antibiotic treatment would not be effective for patients with the R553X mutation, owing to the skipping of exon 11, and further emphasises the need for detailed mechanistic characterisation of the consequences of nonsense disease mutations. PMID:17475917

  12. Dipole entropy based techniques for segmentation of introns and exons in DNA

    NASA Astrophysics Data System (ADS)

    Ramakrishnan, Nithya; Bose, R.

    2012-08-01

    We have used superinformation, which is a measure of the disorder of the entropy content of different portions of a sequence, to analyze the structural variations of the introns and exons in DNA. We have computed superinformation for the angles of the dipole moments of the base-pairs and nucleotides in the double and single-stranded forms of DNA, respectively. We show that the computed dipole-angular superinformation of the introns are significantly higher than those of the exons and that these techniques could be used for intron-exon segmentation. They also yield more accurate and computationally faster results than the previously reported methods.

  13. Rare EGFR exon 18 and exon 20 mutations in non-small-cell lung cancer on 10 117 patients: a multicentre observational study by the French ERMETIC-IFCT network

    PubMed Central

    Beau-Faller, M.; Prim, N.; Ruppert, A.-M.; Nanni-Metéllus, I.; Lacave, R.; Lacroix, L.; Escande, F.; Lizard, S.; Pretet, J.-L.; Rouquette, I.; de Crémoux, P.; Solassol, J.; de Fraipont, F.; Bièche, I.; Cayre, A.; Favre-Guillevin, E.; Tomasini, P.; Wislez, M.; Besse, B.; Legrain, M.; Voegeli, A.-C.; Baudrin, L.; Morin, F.; Zalcman, G.; Quoix, E.; Blons, H.; Cadranel, J.

    2014-01-01

    Background There is scarce data available about epidermal growth factor receptor (EGFR) mutations other than common exon 19 deletions and exon 21 (L858R) mutations. Patients and methods EGFR exon 18 and/or exon 20 mutations were collected from 10 117 non-small-cell lung cancer (NSCLC) samples analysed at 15 French National Cancer Institute (INCa)-platforms of the ERMETIC-IFCT network. Results Between 2008 and 2011, 1047 (10%) samples were EGFR-mutated, 102 (10%) with rare mutations: 41 (4%) in exon 18, 49 (5%) in exon 20, and 12 (1%) with other EGFR mutations. Exon 20 mutations were related to never-smoker status, when compared with exon 18 mutations (P < 0.001). Median overall survival (OS) of metastatic disease was 21 months [95% confidence interval (CI) 12–24], worse in smokers than in non-smoker patients with exon 20 mutations (12 versus 21 months; hazard ratio [HR] for death 0.27, 95% CI 0.08–0.87, P = 0.03). Under EGFR-tyrosine kinase inhibitors (TKIs), median OS was 14 months (95% CI 6–21); disease control rate was better for complex mutations (6 of 7, 86%) than for single mutations (16 of 40, 40%) (P = 0.03). Conclusions Rare EGFR-mutated NSCLCs are heterogeneous, with resistance of distal exon 20 insertions and better sensitivity of exon 18 or complex mutations to EGFR-TKIs, probably requiring individual assessment. PMID:24285021

  14. Gastrointestinal malignancies harbor actionable MET exon 14 deletions

    PubMed Central

    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

  15. Genetic associations of nonsynonymous exonic variants with psychophysiological endophenotypes.

    PubMed

    Vrieze, Scott I; Malone, Stephen M; Pankratz, Nathan; Vaidyanathan, Uma; Miller, Michael B; Kang, Hyun Min; McGue, Matt; Abecasis, Gonçalo; Iacono, William G

    2014-12-01

    We mapped ∼85,000 rare nonsynonymous exonic single nucleotide polymorphisms (SNPs) to 17 psychophysiological endophenotypes in 4,905 individuals, including antisaccade eye movements, resting EEG, P300 amplitude, electrodermal activity, affect-modulated startle eye blink. Nonsynonymous SNPs are predicted to directly change or disrupt proteins encoded by genes and are expected to have significant biological consequences. Most such variants are rare, and new technologies can efficiently assay them on a large scale. We assayed 247,870 mostly rare SNPs on an Illumina exome array. Approximately 85,000 of the SNPs were polymorphic, rare (MAF < .05), and nonsynonymous. Single variant association tests identified a SNP in the PARD3 gene associated with theta resting EEG power. The sequence kernel association test, a gene-based test, identified a gene PNPLA7 associated with pleasant difference startle, the difference in startle magnitude between pleasant and neutral images. No other single nonsynonymous variant, or gene-based group of variants, was strongly associated with any endophenotype. PMID:25387709

  16. Epidermal growth factor receptor exon 20 p.S768I mutation in non-small cell lung carcinoma: A case report combined with a review of the literature and investigation of clinical significance

    PubMed Central

    IMPROTA, GIUSEPPINA; PETTINATO, ANGELA; GIERI, STEFANIA; SCANDURRA, GIUSEPPA; SKOVRIDER-RUMINSKI, WOJCIECH; HØGDALL, ESTRID; FRAGGETTA, FILIPPO

    2016-01-01

    Epidermal growth factor receptor (EGFR) plays a significant role in non-small cell lung cancer (NSCLC), the most prevalent form of lung cancer worldwide. Therefore, EGFR may be a useful molecular target for personalized therapy utilizing tyrosine kinase inhibitors (TKIs). Somatic activating EGFR mutations may be used to identify tumors sensitive to the effects of small-molecule EGFR-TKIs (gefitinib and erlotinib), and alternative, less frequently observed mutations, including the majority of mutations identified within exon 20, may be associated with a lack of response to TKIs. However, due to the comparative rarity of EGFR exon 20 mutations, clinical information concerning the association between EGFR exon 20 mutations and responsiveness to TKIs has been limited within the relevant literature, particularly for certain rare mutations, including p.S768I. The current study reports the case of a patient with NSCLC harboring a p.S768I mutation in the EGFR gene [a substitution at codon 768 of exon 20 (c.2303G>T, p.S768I)], as well as a mutation at codon 719, exon 18 (p.G719A). The relevant literature concerning this rare EGFR somatic mutation is also reviewed. PMID:26870223

  17. De novo exonic mutation in MYH7 gene leading to exon skipping in a patient with early onset muscular weakness and fiber-type disproportion.

    PubMed

    Pajusalu, Sander; Talvik, Inga; Noormets, Klari; Talvik, Tiina; Põder, Haide; Joost, Kairit; Puusepp, Sanna; Piirsoo, Andres; Stenzel, Werner; Goebel, Hans H; Nikopensius, Tiit; Annilo, Tarmo; Nõukas, Margit; Metspalu, Andres; Õunap, Katrin; Reimand, Tiia

    2016-03-01

    Here we report on a case of MYH7-related myopathy in a boy with early onset of muscular weakness and delayed motor development in infancy. His most affected muscles were neck extensors showing a dropped head sign, proximal muscles of lower limbs with positive Gower's sign, and trunk muscles. Brain and spinal cord MRI scans, echocardiography, and laboratory analyses including creatine kinase and lactate did not reveal any abnormalities. Muscle histopathology showed fiber-type disproportion. Whole exome sequencing of the parents-offspring trio revealed a novel de novo c.5655G>A p.(Ala1885=) synonymous substitution of the last nucleotide in exon 38 of the MYH7 gene. Further RNA investigations proved the skipping of exon 38 (p.1854_1885del). This is a first report of an exon-skipping mutation in the MYH7 gene causing myopathy. This report broadens both the phenotypic and genotypic spectra of MYH7-related myopathies. PMID:26782017

  18. 5-hmC in the brain is abundant in synaptic genes and shows differences at the exon-intron boundary

    PubMed Central

    Khare, Tarang; Pai, Shraddha; Koncevicius, Karolis; Pal, Mrinal; Kriukiene, Edita; Liutkeviciute, Zita; Irimia, Manuel; Jia, Peixin; Ptak, Carolyn; Xia, Menghang; Tice, Raymond; Tochigi, Mamoru; Moréra, Solange; Nazarians, Anaies; Belsham, Denise; Wong, Albert H. C.; Blencowe, Benjamin J.; Wang, Sun Chong; Kapranov, Philipp; Kustra, Rafal; Labrie, Viviane; Klimasauskas, Saulius; Petronis, Arturas

    2012-01-01

    5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. Our goal was to characterize the genomic distribution of 5-hmC and 5-mC in human and mouse tissues. We assayed 5-hmC using glucosylation coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in both human and mouse brain. We also identified substantial tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets and with single molecule sequencing. Moreover, in human frontal cortex, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain. PMID:22961382

  19. Alternative splicing of Wilms tumor suppressor 1 (Wt1) exon 4 results in protein isoforms with different functions.

    PubMed

    Schnerwitzki, Danny; Perner, Birgit; Hoppe, Beate; Pietsch, Stefan; Mehringer, Rebecca; Hänel, Frank; Englert, Christoph

    2014-09-01

    The Wilms tumor suppressor gene Wt1 encodes a zinc finger transcription factor that is essential for development of multiple organs including kidneys, gonads, spleen and heart. In mammals Wt1 comprises 10 exons with two characteristic splicing events: inclusion or skipping of exon 5 and alternative usage of two splice donor sites between exons 9 and 10. Most fish including zebrafish and medaka possess two wt1 paralogs, wt1a and wt1b, both lacking exon 5. Here we have characterized wt1 in guppy, platyfish and the short-lived African killifish Nothobranchius furzeri. All fish except zebrafish show alternative splicing of exon 4 of wt1a but not of wt1b with the wt1a(-exon 4) isoform being the predominant splice variant. With regard to function, Wt1a(+exon 4) showed less dimerization but stimulated transcription more effectively than the Wt1a(-exon 4) isoform. A specific knockdown of wt1a exon 4 in zebrafish was associated with anomalies in kidney development demonstrating a physiological function for Wt1a exon 4. Interestingly, alternative splicing of exon 4 seems to be an early evolutionary event as it is observed in the single wt1 gene of the sturgeon, a species that has not gone through teleost-specific genome duplication. PMID:25014653

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

    PubMed

    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. PMID:26703369

  1. A three-dimensional perspective on exon binding by a group II self-splicing intron

    PubMed Central

    Costa, Maria; Michel, François; Westhof, Eric

    2000-01-01

    We have used chemical footprinting, kinetic dissection of reactions and comparative sequence analysis to show that in self-splicing introns belonging to subgroup IIB, the sites that bind the 5′ and 3′ exons are connected to one another by tertiary interactions. This unanticipated arrangement, which contrasts with the direct covalent linkage that prevails in the other major subdivision of group II (subgroup IIA), results in a unique three-dimensional architecture for the complex between the exons, their binding sites and intron domain V. A key feature of the modeled complex is the presence of several close contacts between domain V and one of the intron–exon pairings. These contacts, whose existence is supported by hydroxyl radical footprinting, provide a structural framework for the known role of domain V in catalysis and its recently demonstrated involvement in binding of the 5′ exon. PMID:10990464

  2. Exon array data analysis using Affymetrix power tools and R statistical software

    PubMed Central

    2011-01-01

    The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform. PMID:21498550

  3. Recurring exon deletions in the haptoglobin (HP) gene associate with lower blood cholesterol levels

    PubMed Central

    Boettger, Linda M.; Salem, Rany M.; Handsaker, Robert E.; Peloso, Gina; Kathiresan, Sekar; Hirschhorn, Joel; McCarroll, Steven A.

    2016-01-01

    Two exons of the human haptoglobin (HP) gene exhibit copy number variation that affects HP multimerization and underlies one of the first protein polymorphisms identified in humans. The evolutionary origins and medical significance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from the recurring reversion of an ancient hominin-specific duplication of these exons. Though this polymorphism has been largely invisible to genome-wide genetic studies to date, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We show that these deletions, and a SNP that affects HP expression, are the likely drivers of the strong but complex association of cholesterol levels to SNPs near HP. Recurring exonic deletions in the haptoglobin gene likely enhance human health by lowering cholesterol levels in the blood. PMID:26901066

  4. Identification of intron/exon boundaries in genomic DNA by inverse PCR.

    PubMed

    Albertsen, H; Thliveris, A

    2001-05-01

    This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. Cloned genomic DNA is prepared for inverse polymerase chain reaction (PCR) by digesting the DNA with a restriction enzyme and circularizing the restriction fragments by ligation. Diverging primer pairs for each exon are designed on the basis of the cDNA sequence. The circularized restriction fragments are amplified using these diverging primers, the PCR product is sequenced, and the sequence is compared to the cDNA sequence to determine the location of the intron/exon boundaries. The lower complexity of cloned DNA (e.g., YAC, P1, or cosmid DNA) facilitates preparation of good template. This unit describes identifying intron/exon boundaries in genomic DNA by comparing nucleotide sequences of genomic DNA to cDNA. PMID:18428300

  5. Recurring exon deletions in the HP (haptoglobin) gene contribute to lower blood cholesterol levels.

    PubMed

    Boettger, Linda M; Salem, Rany M; Handsaker, Robert E; Peloso, Gina M; Kathiresan, Sekar; Hirschhorn, Joel N; McCarroll, Steven A

    2016-04-01

    One of the first protein polymorphisms identified in humans involves the abundant blood protein haptoglobin. Two exons of the HP gene (encoding haptoglobin) exhibit copy number variation that affects HP protein structure and multimerization. The evolutionary origins and medical relevance of this polymorphism have been uncertain. Here we show that this variation has likely arisen from many recurring deletions, more specifically, reversions of an ancient hominin-specific duplication of these exons. Although this polymorphism has been largely invisible to genome-wide genetic studies thus far, we describe a way to analyze it by imputation from SNP haplotypes and find among 22,288 individuals that these HP exonic deletions associate with reduced LDL and total cholesterol levels. We further show that these deletions, and a SNP that affects HP expression, appear to drive the strong association of cholesterol levels with SNPs near HP. Recurring exonic deletions in HP likely enhance human health by lowering cholesterol levels in the blood. PMID:26901066

  6. Nanoparticle Delivery of Antisense Oligonucleotides and Their Application in the Exon Skipping Strategy for Duchenne Muscular Dystrophy

    PubMed Central

    Falzarano, Maria Sofia; Passarelli, Chiara

    2014-01-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy. PMID:24506782

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

    SciTech Connect

    Tsai, A.C.; Allingham-Hawkins, D.J.; Becker, L.

    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.

  8. Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations

    PubMed Central

    Dröge, Carola; Schaal, Heiner; Engelmann, Guido; Wenning, Daniel; Häussinger, Dieter; Kubitz, Ralf

    2016-01-01

    The bile salt export pump BSEP mediates bile formation. Over 150 BSEP mutations are associated with progressive familial intrahepatic cholestasis type 2 (PFIC-2), with few characterised specifically. We examined liver tissues from two PFIC-2 patients compound heterozygous for the splice-site mutation c.150 + 3A > C and either c.2783_2787dup5 resulting in a frameshift with a premature termination codon (child 1) or p.R832C (child 2). Splicing was analysed with a minigene system and mRNA sequencing from patients’ livers. Protein expression was shown by immunofluorescence. Using the minigene, c.150 + 3A > C causes complete skipping of exon 3. In liver tissue of child 1, c.2783_2787dup5 was found on DNA but not on mRNA level, implying nonsense-mediated mRNA decay (NMD) when c.2783_2787dup5 is present. Still, BSEP protein as well as mRNA with and without exon 3 were detectable and can be assigned to the c.150 + 3A > C allele. Correctly spliced transcripts despite c.150 + 3A > C were also confirmed in liver of child 2. In conclusion, we provide evidence (1) for effective NMD due to a BSEP frameshift mutation and (2) partial exon-skipping due to c.150 + 3A > C. The results illustrate that the extent of exon-skipping depends on the genomic and cellular context and that regulation of splicing may have therapeutic potential. PMID:27114171

  9. Exon-skipping and mRNA decay in human liver tissue: molecular consequences of pathogenic bile salt export pump mutations.

    PubMed

    Dröge, Carola; Schaal, Heiner; Engelmann, Guido; Wenning, Daniel; Häussinger, Dieter; Kubitz, Ralf

    2016-01-01

    The bile salt export pump BSEP mediates bile formation. Over 150 BSEP mutations are associated with progressive familial intrahepatic cholestasis type 2 (PFIC-2), with few characterised specifically. We examined liver tissues from two PFIC-2 patients compound heterozygous for the splice-site mutation c.150 + 3A > C and either c.2783_2787dup5 resulting in a frameshift with a premature termination codon (child 1) or p.R832C (child 2). Splicing was analysed with a minigene system and mRNA sequencing from patients' livers. Protein expression was shown by immunofluorescence. Using the minigene, c.150 + 3A > C causes complete skipping of exon 3. In liver tissue of child 1, c.2783_2787dup5 was found on DNA but not on mRNA level, implying nonsense-mediated mRNA decay (NMD) when c.2783_2787dup5 is present. Still, BSEP protein as well as mRNA with and without exon 3 were detectable and can be assigned to the c.150 + 3A > C allele. Correctly spliced transcripts despite c.150 + 3A > C were also confirmed in liver of child 2. In conclusion, we provide evidence (1) for effective NMD due to a BSEP frameshift mutation and (2) partial exon-skipping due to c.150 + 3A > C. The results illustrate that the extent of exon-skipping depends on the genomic and cellular context and that regulation of splicing may have therapeutic potential. PMID:27114171

  10. Computer analysis of protein functional sites projection on exon structure of genes in Metazoa

    PubMed Central

    2015-01-01

    Background Study of the relationship between the structural and functional organization of proteins and their coding genes is necessary for an understanding of the evolution of molecular systems and can provide new knowledge for many applications for designing proteins with improved medical and biological properties. It is well known that the functional properties of proteins are determined by their functional sites. Functional sites are usually represented by a small number of amino acid residues that are distantly located from each other in the amino acid sequence. They are highly conserved within their functional group and vary significantly in structure between such groups. According to this facts analysis of the general properties of the structural organization of the functional sites at the protein level and, at the level of exon-intron structure of the coding gene is still an actual problem. Results One approach to this analysis is the projection of amino acid residue positions of the functional sites along with the exon boundaries to the gene structure. In this paper, we examined the discontinuity of the functional sites in the exon-intron structure of genes and the distribution of lengths and phases of the functional site encoding exons in vertebrate genes. We have shown that the DNA fragments coding the functional sites were in the same exons, or in close exons. The observed tendency to cluster the exons that code functional sites which could be considered as the unit of protein evolution. We studied the characteristics of the structure of the exon boundaries that code, and do not code, functional sites in 11 Metazoa species. This is accompanied by a reduced frequency of intercodon gaps (phase 0) in exons encoding the amino acid residue functional site, which may be evidence of the existence of evolutionary limitations to the exon shuffling. Conclusions These results characterize the features of the coding exon-intron structure that affect the

  11. PVT1 Exon 9: A Potential Biomarker of Aggressive Prostate Cancer?

    PubMed Central

    Ilboudo, Adeodat; Chouhan, Jyoti; McNeil, Brian K.; Osborne, Joseph R.; Ogunwobi, Olorunseun O.

    2015-01-01

    Prostate cancer (PCa) is the most commonly diagnosed cancer as well as the greatest source of cancer-related mortality in males of African ancestry (MoAA). Interestingly, this has been shown to be associated with single nucleotide polymorphisms around regions 2 and 3 of the 8q24 human chromosomal region. The non-protein coding gene locus Plasmacytoma Variant Translocation 1 (PVT1) is located at 8q24 and is overexpressed in PCa and, therefore, is also a candidate biomarker to explain the well-known disparity in this group. PVT1 has at least 12 exons that make separate transcripts which may have different functions, all of which are at present unknown in PCa. Our aim was to determine if any PVT1 transcripts play a role in aggressiveness and racial disparity in PCa. We used a panel of seven PCa cell lines including three derived from MoAA. Ribonucleic acid extraction, complementary deoxyribonucleic acid synthesis, and quantitative polymerase chain reaction (qPCR) were performed to evaluate expression of all 12 PVT1 exons. Each qPCR was performed in quadruplicates. At least four separate qPCR experiments were performed. Expression of PVT1 exons was inconsistent except for exon 9. There was no significant difference in exon 9 expression between cell lines derived from Caucasian males (CM), and an indolent cell line derived from MoAA. However, exon 9 expression in the aggressive MDA PCa 2b and E006AA-hT cell lines derived from MoAA was significantly higher than in other cell lines. Consequently, we observed differential expression of exon 9 of PVT1 in a manner that suggests that PVT1 exon 9 may be associated with aggressive PCa in MoAA. PMID:26703666

  12. Evolutionary connections between coding and splicing regulatory regions in the fibronectin EDA exon.

    PubMed

    Zago, Paola; Buratti, Emanuele; Stuani, Cristiana; Baralle, Francisco E

    2011-08-01

    Research on exonic coding sequences has demonstrated that many substitutions at the amino acid level may also reflect profound changes at the level of splicing regulatory regions. These results have revealed that, for many alternatively spliced exons, there is considerable pressure to strike a balance between two different and sometimes conflicting forces: the drive to improve the quality and production efficiency of proteins and the maintenance of proper exon recognition by the splicing machinery. Up to now, the systems used to investigate these connections have mostly focused on short alternatively spliced exons that contain a high density of splicing regulatory elements. Although this is obviously a desirable feature in order to maximize the chances of spotting connections, it also complicates the process of drawing straightforward evolutionary pathways between different species (because of the numerous alternative pathways through which the same end point can be achieved). The alternatively spliced fibronectin extra domain A exon (also referred to as EDI or EIIIA) does not have these limitations, as its inclusion is already known to depend on a single exonic splicing enhancer element within its sequence. In this study, we have compared the rat and human fibronectin EDA exons with regard to RNA structure, exonic splicing enhancer strengths, and SR protein occupancy. The results gained from these analyses have then been used to perform an accurate evaluation of EDA sequences observed in a wide range of animal species. This comparison strongly suggests the existence of an evolutionary connection between changes at the nucleotide levels and the need to maintain efficient EDA recognition in different species. PMID:21663748

  13. Identification of a new class of exonic splicing enhancers by in vivo selection.

    PubMed Central

    Coulter, L R; Landree, M A; Cooper, T A

    1997-01-01

    In vitro selection strategies have typically been used to identify a preferred ligand, usually an RNA, for an identified protein. Ideally, one would like to know RNA consensus sequences preferred in vivo for as-yet-unidentified factors. The ability to select RNA-processing signals would be particularly beneficial in the analysis of exon enhancer sequences that function in exon recognition during pre-mRNA splicing. Exon enhancers represent a class of potentially ubiquitous RNA-processing signals whose actual prevalence is unknown. To establish an approach for in vivo selection, we developed an iterative scheme to select for exon sequences that enhance exon inclusion. This approach is modeled on the in vitro SELEX procedure and uses transient transfection in an iterative procedure to enrich RNA-processing signals in cultured vertebrate cells. Two predominant sequence motifs were enriched after three rounds of selection: a purine-rich motif that resembles previously identified splicing enhancers and a class of A/C-rich splicing enhancers (ACEs). Individual selected ACEs enhanced splicing in vivo and in vitro. ACE splicing activity was competed by RNAs containing the purine-rich splicing enhancer from cardiac troponin T exon 5. Thus, ACE activity is likely to require a subset of the SR splicing factors previously shown to mediate activity of this purine-rich enhancer. ACE motifs are found in two vertebrate exons previously demonstrated to contain splicing enhancer activity as well as in the well-characterized Drosophila doublesex (dsx) splicing enhancer. We demonstrate that one copy of the dsx repeat enhances splicing of a vertebrate exon in vertebrate cells and that this enhancer activity requires the ACE motif. We suggest the possibility that the dsx enhancer is a member of a previously unrecognized family of ACEs. PMID:9121463

  14. Exon size affects competition between splicing and cleavage-polyadenylation in the immunoglobulin mu gene.

    PubMed

    Peterson, M L; Bryman, M B; Peiter, M; Cowan, C

    1994-01-01

    The alternative RNA processing of microseconds and microns mRNAs from a single primary transcript depends on competition between a cleavage-polyadenylation reaction to produce microseconds mRNA and a splicing reaction to produce microns mRNA. The ratio of microseconds to microns mRNA is regulated during B-cell maturation; relatively more spliced microns mRNA is made in B cells than in plasma cells. The balance between the efficiencies of splicing and cleavage-polyadenylation is critical to the regulation. The mu gene can be modified to either reduce or improve the efficiency of each reaction and thus alter the ratio of the two RNAs produced. However, as long as neither reaction is so strong that it totally dominates, expression of the modified mu genes is regulated in B cells and plasma cells. The current experiments reveal a relationship between the C mu 4 exon size and the microseconds/microns expression ratio. The shorter the distance between the C mu 4 5' splice site and the nearest upstream 3' splice site, the more spliced microns mRNA was produced. Conversely, when this exon was expanded, more microseconds mRNA was produced. Expression from these mu genes with altered exon sizes were regulated between B cells and plasma cells. Since RNA processing in the mu gene can be considered a competition between defining the C mu 4 exon as an internal exon (in microns mRNA) versus a terminal exon (in microseconds mRNA), exon size may affect the competition among factors interacting with this exon. PMID:7903422

  15. Further genotype-phenotype correlation emerging from two families with PLP1 exon 4 skipping.

    PubMed

    Biancheri, Roberta; Grossi, Serena; Regis, Stefano; Rossi, Andrea; Corsolini, Fabio; Rossi, Daniela Paola; Cavalli, Pietro; Severino, Mariasavina; Filocamo, Mirella

    2014-03-01

    Proteolipid protein 1 (PLP1) gene-related disorders due to mutations in the PLP1 include a wide spectrum of X-linked disorders ranging from severe connatal Pelizaeus-Merzbacher disease (PMD) to spastic paraplegia 2 (SPG2). Duplications, deletions or point mutations in coding and noncoding regions of the PLP1 gene may occur. We report the clinical, neuroradiologic and molecular findings in six patients from two unrelated families. The affected males showed severe mental retardation, spastic tetraparesis, inability of walking and pes cavus at onset in early infancy. Brain magnetic resonance imaging (MRI) showed hypomyelination and brain atrophy. Nystagmus was never observed. The affected females showed adult-onset progressive spastic paraparesis leading to wheel-chair dependency and subtle white matter changes on brain MRI. Molecular studies in the two families identified two different intronic mutations, the novel c.622+2T>C and the known c.622+1G>A, leading to the skipping of PLP1-exon 4. The clinical presentation of the affected males did not consistently fit in any of the PLP1-related disorder subtypes (i.e., connatal or classic PMD, SPG2 and 'PLP1 null syndrome'), and in addition, the carrier females were symptomatic despite the severe clinical picture of their respective probands. This study provides new insight into the genotype-phenotype correlations of patients with PLP1 splice-site mutations. PMID:23711321

  16. Tight regulation of plant immune responses by combining promoter and suicide exon elements.

    PubMed

    Gonzalez, Tania L; Liang, Yan; Nguyen, Bao N; Staskawicz, Brian J; Loqué, Dominique; Hammond, Ming C

    2015-08-18

    Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive 'hypersensitive response' (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes. PMID:26138488

  17. Tight regulation of plant immune responses by combining promoter and suicide exon elements

    SciTech Connect

    Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.; Staskawicz, Brian J.; Loqué, Dominique; Hammond, Ming C.

    2015-07-02

    Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. In conclusion, beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.

  18. Tight regulation of plant immune responses by combining promoter and suicide exon elements

    DOE PAGESBeta

    Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.; Staskawicz, Brian J.; Loqué, Dominique; Hammond, Ming C.

    2015-07-02

    Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightlymore » regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. In conclusion, beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes.« less

  19. Tight regulation of plant immune responses by combining promoter and suicide exon elements

    PubMed Central

    Gonzalez, Tania L.; Liang, Yan; Nguyen, Bao N.; Staskawicz, Brian J.; Loqué, Dominique; Hammond, Ming C.

    2015-01-01

    Effector-triggered immunity (ETI) is activated when plant disease resistance (R) proteins recognize the presence of pathogen effector proteins delivered into host cells. The ETI response generally encompasses a defensive ‘hypersensitive response’ (HR) that involves programmed cell death at the site of pathogen recognition. While many R protein and effector protein pairs are known to trigger HR, other components of the ETI signaling pathway remain elusive. Effector genes regulated by inducible promoters cause background HR due to leaky protein expression, preventing the generation of relevant transgenic plant lines. By employing the HyP5SM suicide exon, we have developed a strategy to tightly regulate effector proteins such that HR is chemically inducible and non-leaky. This alternative splicing-based gene regulation system was shown to successfully control Bs2/AvrBs2-dependent and RPP1/ATR1Δ51-dependent HR in Nicotiana benthamiana and Nicotiana tabacum, respectively. It was also used to generate viable and healthy transgenic Arabidopsis thaliana plants that inducibly initiate HR. Beyond enabling studies on the ETI pathway, our regulatory strategy is generally applicable to reduce or eliminate undesired background expression of transgenes. PMID:26138488

  20. Loss of exon identity is a common mechanism of human inherited disease

    PubMed Central

    Sterne-Weiler, Timothy; Howard, Jonathan; Mort, Matthew; Cooper, David N.; Sanford, Jeremy R.

    2011-01-01

    It is widely accepted that at least 10% of all mutations causing human inherited disease disrupt splice-site consensus sequences. In contrast to splice-site mutations, the role of auxiliary cis-acting elements such as exonic splicing enhancers (ESE) and exonic splicing silencers (ESS) in human inherited disease is still poorly understood. Here we use a top-down approach to determine rates of loss or gain of known human exonic splicing regulatory (ESR) sequences associated with either disease-causing mutations or putatively neutral single nucleotide polymorphisms (SNPs). We observe significant enrichment toward loss of ESEs and gain of ESSs among inherited disease-causing variants relative to neutral polymorphisms, indicating that exon skipping may play a prominent role in aberrant gene regulation. Both computational and biochemical approaches underscore the relevance of exonic splicing enhancer loss and silencer gain in inherited disease. Additionally, we provide direct evidence that both SRp20 (SRSF3) and possibly PTB (PTBP1) are involved in the function of a splicing silencer that is created de novo by a total of 83 different inherited disease mutations in 67 different disease genes. Taken together, we find that ∼25% (7154/27,681) of known mis-sense and nonsense disease-causing mutations alter functional splicing signals within exons, suggesting a much more widespread role for aberrant mRNA processing in causing human inherited disease than has hitherto been appreciated. PMID:21750108

  1. Molecular characterization of exon 3 of caprine myostatin gene in Marwari goat

    PubMed Central

    Khichar, Jai Prakash; Gahlot, Gyan Chand; Agrawal, Vijay Kumar; Kiran; Dewna, Ajay Singh; Prakash; Ashraf, Mohammad

    2016-01-01

    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. PMID:27397994

  2. Single exon-resolution targeted chromosomal microarray analysis of known and candidate intellectual disability genes.

    PubMed

    Tucker, Tracy; Zahir, Farah R; Griffith, Malachi; Delaney, Allen; Chai, David; Tsang, Erica; Lemyre, Emmanuelle; Dobrzeniecka, Sylvia; Marra, Marco; Eydoux, Patrice; Langlois, Sylvie; Hamdan, Fadi F; Michaud, Jacques L; Friedman, Jan M

    2014-06-01

    Intellectual disability affects about 3% of individuals globally, with∼50% idiopathic. We designed an exonic-resolution array targeting all known submicroscopic chromosomal intellectual disability syndrome loci, causative genes for intellectual disability, and potential candidate genes, all genes encoding glutamate receptors and epigenetic regulators. Using this platform, we performed chromosomal microarray analysis on 165 intellectual disability trios (affected child and both normal parents). We identified and independently validated 36 de novo copy-number changes in 32 trios. In all, 67% of the validated events were intragenic, involving only exon 1 (which includes the promoter sequence according to our design), exon 1 and adjacent exons, or one or more exons excluding exon 1. Seventeen of the 36 copy-number variants involve genes known to cause intellectual disability. Eleven of these, including seven intragenic variants, are clearly pathogenic (involving STXBP1, SHANK3 (3 patients), IL1RAPL1, UBE2A, NRXN1, MEF2C, CHD7, 15q24 and 9p24 microdeletion), two are likely pathogenic (PI4KA, DCX), two are unlikely to be pathogenic (GRIK2, FREM2), and two are unclear (ARID1B, 15q22 microdeletion). Twelve individuals with genomic imbalances identified by our array were tested with a clinical microarray, and six had a normal result. We identified de novo copy-number variants within genes not previously implicated in intellectual disability and uncovered pathogenic variation of known intellectual disability genes below the detection limit of standard clinical diagnostic chromosomal microarray analysis. PMID:24253858

  3. Spectrum of splicing errors caused by CHRNE mutations affecting introns and intron/exon boundaries

    PubMed Central

    Ohno, K; Tsujino, A; Shen, X; Milone, M; Engel, A

    2005-01-01

    Background: Mutations in CHRNE, the gene encoding the muscle nicotinic acetylcholine receptor ε subunit, cause congenital myasthenic syndromes. Only three of the eight intronic splice site mutations of CHRNE reported to date have had their splicing consequences characterised. Methods: We analysed four previously reported and five novel splicing mutations in CHRNE by introducing the entire normal and mutant genomic CHRNEs into COS cells. Results and conclusions: We found that short introns (82–109 nucleotides) favour intron retention, whereas medium to long introns (306–1210 nucleotides) flanking either or both sides of an exon favour exon skipping. Two mutations are of particular interest. Firstly, a G→T substitution at the 3' end of exon 8 predicts an R286M missense mutation, but instead results in skipping of exon 8. In human genes, a mismatch of the last exonic nucleotide to U1 snRNP is frequently compensated by a matching nucleotide at intron position +6. CHRNE intron 8 has a mismatch at position +6, and accordingly fails to compensate for the exonic mutation at position –1. Secondly, a 16 bp duplication, giving rise to two 3' splice sites (g.IVS10-9_c.1167dup16), results in silencing of the downstream 3' splice site. This conforms to the scanning model of recognition of the 3' splice site, which predicts that the first "ag" occurring after the branch point is selected for splicing. PMID:16061559

  4. Conservation of CD44 exon v3 functional elements in mammals

    PubMed Central

    Vela, Elena; Hilari, Josep M; Delclaux, María; Fernández-Bellon, Hugo; Isamat, Marcos

    2008-01-01

    Background The human CD44 gene contains 10 variable exons (v1 to v10) that can be alternatively spliced to generate hundreds of different CD44 protein isoforms. Human CD44 variable exon v3 inclusion in the final mRNA depends on a multisite bipartite splicing enhancer located within the exon itself, which we have recently described, and provides the protein domain responsible for growth factor binding to CD44. Findings We have analyzed the sequence of CD44v3 in 95 mammalian species to report high conservation levels for both its splicing regulatory elements (the 3' splice site and the exonic splicing enhancer), and the functional glycosaminglycan binding site coded by v3. We also report the functional expression of CD44v3 isoforms in peripheral blood cells of different mammalian taxa with both consensus and variant v3 sequences. Conclusion CD44v3 mammalian sequences maintain all functional splicing regulatory elements as well as the GAG binding site with the same relative positions and sequence identity previously described during alternative splicing of human CD44. The sequence within the GAG attachment site, which in turn contains the Y motif of the exonic splicing enhancer, is more conserved relative to the rest of exon. Amplification of CD44v3 sequence from mammalian species but not from birds, fish or reptiles, may lead to classify CD44v3 as an exclusive mammalian gene trait. PMID:18710510

  5. Antisense-mediated exon skipping: A versatile tool with therapeutic and research applications

    PubMed Central

    Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B.

    2007-01-01

    Antisense-mediated modulation of splicing is one of the few fields where antisense oligonucleotides (AONs) have been able to live up to their expectations. In this approach, AONs are implemented to restore cryptic splicing, to change levels of alternatively spliced genes, or, in case of Duchenne muscular dystrophy (DMD), to skip an exon in order to restore a disrupted reading frame. The latter allows the generation of internally deleted, but largely functional, dystrophin proteins and would convert a severe DMD into a milder Becker muscular dystrophy phenotype. In fact, exon skipping is currently one of the most promising therapeutic tools for DMD, and a successful first-in-man trial has recently been completed. In this review the applicability of exon skipping for DMD and other diseases is described. For DMD AONs have been designed for numerous exons, which has given us insight into their mode of action, splicing in general, and splicing of the DMD gene in particular. In addition, retrospective analysis resulted in guidelines for AON design for DMD and most likely other genes as well. This knowledge allows us to optimize therapeutic exon skipping, but also opens up a range of other applications for the exon skipping approach. PMID:17684229

  6. Evolution of the Exon-Intron Structure in Ciliate Genomes.

    PubMed

    Bondarenko, Vladyslav S; Gelfand, Mikhail S

    2016-01-01

    A typical eukaryotic gene is comprised of alternating stretches of regions, exons and introns, retained in and spliced out a mature mRNA, respectively. Although the length of introns may vary substantially among organisms, a large fraction of genes contains short introns in many species. Notably, some Ciliates (Paramecium and Nyctotherus) possess only ultra-short introns, around 25 bp long. In Paramecium, ultra-short introns with length divisible by three (3n) are under strong evolutionary pressure and have a high frequency of in-frame stop codons, which, in the case of intron retention, cause premature termination of mRNA translation and consequent degradation of the mis-spliced mRNA by the nonsense-mediated decay mechanism. Here, we analyzed introns in five genera of Ciliates, Paramecium, Tetrahymena, Ichthyophthirius, Oxytricha, and Stylonychia. Introns can be classified into two length classes in Tetrahymena and Ichthyophthirius (with means 48 bp, 69 bp, and 55 bp, 64 bp, respectively), but, surprisingly, comprise three distinct length classes in Oxytricha and Stylonychia (with means 33-35 bp, 47-51 bp, and 78-80 bp). In most ranges of the intron lengths, 3n introns are underrepresented and have a high frequency of in-frame stop codons in all studied species. Introns of Paramecium, Tetrahymena, and Ichthyophthirius are preferentially located at the 5' and 3' ends of genes, whereas introns of Oxytricha and Stylonychia are strongly skewed towards the 5' end. Analysis of evolutionary conservation shows that, in each studied genome, a significant fraction of intron positions is conserved between the orthologs, but intron lengths are not correlated between the species. In summary, our study provides a detailed characterization of introns in several genera of Ciliates and highlights some of their distinctive properties, which, together, indicate that splicing spellchecking is a universal and evolutionarily conserved process in the biogenesis of short introns in

  7. RBFOX and PTBP1 proteins regulate the alternative splicing of micro-exons in human brain transcripts

    PubMed Central

    Sanchez-Pulido, Luis; Haerty, Wilfried

    2015-01-01

    Ninety-four percent of mammalian protein-coding exons exceed 51 nucleotides (nt) in length. The paucity of micro-exons (≤ 51 nt) suggests that their recognition and correct processing by the splicing machinery present greater challenges than for longer exons. Yet, because thousands of human genes harbor processed micro-exons, specialized mechanisms may be in place to promote their splicing. Here, we survey deep genomic data sets to define 13,085 micro-exons and to study their splicing mechanisms and molecular functions. More than 60% of annotated human micro-exons exhibit a high level of sequence conservation, an indicator of functionality. While most human micro-exons require splicing-enhancing genomic features to be processed, the splicing of hundreds of micro-exons is enhanced by the adjacent binding of splice factors in the introns of pre-messenger RNAs. Notably, splicing of a significant number of micro-exons was found to be facilitated by the binding of RBFOX proteins, which promote their inclusion in the brain, muscle, and heart. Our analyses suggest that accurate regulation of micro-exon inclusion by RBFOX proteins and PTBP1 plays an important role in the maintenance of tissue-specific protein–protein interactions. PMID:25524026

  8. Systematic Dissection of Coding Exons at Single Nucleotide Resolution Supports an Additional Role in Cell-Specific Transcriptional Regulation

    PubMed Central

    Kim, Mee J.; Findlay, Gregory M.; Martin, Beth; Zhao, Jingjing; Bell, Robert J. A.; Smith, Robin P.; Ku, Angel A.; Shendure, Jay; Ahituv, Nadav

    2014-01-01

    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. PMID:25340400

  9. JAK2 Exon 14 Deletion in Patients with Chronic Myeloproliferative Neoplasms

    PubMed Central

    Ma, Wanlong; Kantarjian, Hagop; Zhang, Xi; Wang, Xiuqiang; Zhang, Zhong; Yeh, Chen-Hsiung; O'Brien, Susan; Giles, Francis; Bruey, Jean Marie; Albitar, Maher

    2010-01-01

    Background The JAK2 V617F mutation in exon 14 is the most common mutation in chronic myeloproliferative neoplasms (MPNs); deletion of the entire exon 14 is rarely detected. In our previous study of >10,000 samples from patients with suspected MPNs tested for JAK2 mutations by reverse transcription-PCR (RT-PCR) with direct sequencing, complete deletion of exon 14 (Δexon14) constituted <1% of JAK2 mutations. This appears to be an alternative splicing mutation, not detectable with DNA-based testing. Methodology/Principal Findings We investigated the possibility that MPN patients may express the JAK2 Δexon14 at low levels (<15% of total transcript) not routinely detectable by RT-PCR with direct sequencing. Using a sensitive RT-PCR–based fluorescent fragment analysis method to quantify JAK2 Δexon14 mRNA expression relative to wild-type, we tested 61 patients with confirmed MPNs, 183 with suspected MPNs (93 V617F-positive, 90 V617F-negative), and 46 healthy control subjects. The Δexon14 variant was detected in 9 of the 61 (15%) confirmed MPN patients, accounting for 3.96% to 33.85% (mean  = 12.04%) of total JAK2 transcript. This variant was also detected in 51 of the 183 patients with suspected MPNs (27%), including 20 of the 93 (22%) with V617F (mean [range] expression  = 5.41% [2.13%–26.22%]) and 31 of the 90 (34%) without V617F (mean [range] expression  = 3.88% [2.08%–12.22%]). Immunoprecipitation studies demonstrated that patients expressing Δexon14 mRNA expressed a corresponding truncated JAK2 protein. The Δexon14 variant was not detected in the 46 control subjects. Conclusions/Significance These data suggest that expression of the JAK2 Δexon14 splice variant, leading to a truncated JAK2 protein, is common in patients with MPNs. This alternatively spliced transcript appears to be more frequent in MPN patients without V617F mutation, in whom it might contribute to leukemogenesis. This mutation is missed if DNA rather than RNA is used for

  10. Molecular basis of recessive congenital methemoglobinemia, types I and II: Exon skipping and three novel missense mutations in the NADH-cytochrome b5 reductase (diaphorase 1) gene.

    PubMed

    Kugler, W; Pekrun, A; Laspe, P; Erdlenbruch, B; Lakomek, M

    2001-04-01

    Hereditary methemoglobinemia due to reduced nicotin amide adenine dinucleotide (NADH)-cytochrome b5 reductase (b5r) deficiency is classified into an erythrocyte type (I) and a generalized type (II). We investigated the b5r gene of three unrelated patients with types I and II and found four novel mutations. The patient with type I was homozygous for a c.535 G-->A exchange in exon 6 (A179T). The patients with type II were found to be homozygous for a c.757 G-->A transition in exon 9 (V253M) and compound heterozygous for two mutations, respectively. One allele presented a c.379 A-->G transition (M127V). The second allele carried a sequence difference at the invariant 3' splice-acceptor dinucleotide of intron 4 (IVS4-2A-->G) resulting in skipping of exon 5. To characterize a possible effect of this mutation on RNA metabolism, poly(A)(+) RNA was analyzed by RT-PCR and sequencing. The results show that RNA is made from the allele harboring the 3'-splice site mutation. Furthermore, western blot analysis revealed a complete absence of immunologically detectable b5r in skin fibroblasts of this patient. The compound heterozygosity for the splice site and the missense mutations apparently caused hereditary methemoglobinemia type II in this patient. Hum Mutat 17:348, 2001. PMID:11295830

  11. Identification of Novel Protein–Ligand Interactions by Exon Microarray Analysis of Yeast Surface Displayed cDNA Library Selection Outputs

    PubMed Central

    Bidlingmaier, Scott; Liu, Bin

    2016-01-01

    Yeast surface display is widely utilized to screen large libraries for proteins or protein fragments with specific binding properties. We have previously constructed and utilized yeast surface displayed human cDNA libraries to identify protein fragments that bind to various target ligands. Conventional approaches employ monoclonal screening and sequencing of polyclonal outputs that have been enriched for binding to a target molecule by several rounds of affinity-based selection. Frequently, a small number of clones will dominate the selection output, making it difficult to comprehensively identify potentially important interactions due to low representation in the selection output. We have developed a novel method to address this problem. By analyzing selection outputs using high-density human exon microarrays, the full potential of selection output diversity can be revealed in one experiment. FACS-based selection using yeast surface displayed human cDNA libraries combined with exon microarray analysis of the selection outputs is a powerful way of rapidly identifying protein fragments with affinity for any soluble ligand that can be fluorescently detected, including small biological molecules and drugs. In this report we present protocols for exon microarray-based analysis of yeast surface display human cDNA library selection outputs. PMID:26060075

  12. Identification of a novel, recurrent HEY1-NCOA2 fusion in mesenchymal chondrosarcoma based on a genome-wide screen of exon-level expression data.

    PubMed

    Wang, Lu; Motoi, Toru; Khanin, Raya; Olshen, Adam; Mertens, Fredrik; Bridge, Julia; Dal Cin, Paola; Antonescu, Cristina R; Singer, Samuel; Hameed, Meera; Bovee, Judith V M G; Hogendoorn, Pancras C W; Socci, Nicholas; Ladanyi, Marc

    2012-02-01

    Cancer gene fusions that encode a chimeric protein are often characterized by an intragenic discontinuity in the RNA\\expression levels of the exons that are 5' or 3' to the fusion point in one or both of the fusion partners due to differences in the levels of activation of their respective promoters. Based on this, we developed an unbiased, genome-wide bioinformatic screen for gene fusions using Affymetrix Exon array expression data. Using a training set of 46 samples with different known gene fusions, we developed a data analysis pipeline, the "Fusion Score (FS) model", to score and rank genes for intragenic changes in expression. In a separate discovery set of 41 tumor samples with possible unknown gene fusions, the FS model generated a list of 552 candidate genes. The transcription factor gene NCOA2 was one of the candidates identified in a mesenchymal chondrosarcoma. A novel HEY1-NCOA2 fusion was identified by 5' RACE, representing an in-frame fusion of HEY1 exon 4 to NCOA2 exon 13. RT-PCR or FISH evidence of this HEY1-NCOA2 fusion was present in all additional mesenchymal chondrosarcomas tested with a definitive histologic diagnosis and adequate material for analysis (n = 9) but was absent in 15 samples of other subtypes of chondrosarcomas. We also identified a NUP107-LGR5 fusion in a dedifferentiated liposarcoma but analysis of 17 additional samples did not confirm it as a recurrent event in this sarcoma type. The novel HEY1-NCOA2 fusion appears to be the defining and diagnostic gene fusion in mesenchymal chondrosarcomas. PMID:22034177

  13. Mutation Screening of Exons 7 and 13 of the TMC1 Gene in Autosomal Recessive Non-syndromic Hearing Loss (ARNSHL) in Iran

    PubMed Central

    Moradipour, Negar; Ghasemi-Dehkordi, Payam; Heibati, Fatemeh; Parchami-Barjui, Shahrbanuo; Abolhasani, Marziyeh; Rashki, Ahmad; Hashemzadeh-Chaleshtori, Morteza

    2016-01-01

    Background: Non-syndromic hearing loss (NSHL) is the most common birth defect and occurs in approximately 1/1,000 newborns. NSHL is a heterogeneous trait and can arise due to both genetic and environmental factors. Mutations of the transmembrane channel-like 1 (TMC1) gene cause non-syndromic deafness in humans and mice. Objectives: The aim of the present study was to investigate the association of TMC1 gene mutations of the locus DFNB7/11 in exons 7 and 13 in a cohort of 100 patients with hearing loss in Iran using polymerase chain reaction–single-stranded conformation polymorphism (PCR-SSCP), heteroduplex analysis (HA), and DNA sequencing. Patients and Methods: In this experimental study, the blood samples of 100 NSHL patients were collected from 10 provinces in Iran. These patients had a mean age of 16.5 ± 2.01 years and 74.15% of their parents had consanguinity. DNA was extracted from specimens and mutations of exons 7 and 13 of the TMC1 gene were investigated using PCR-SSCP. All samples were checked via HA reaction and suspected specimens with shift bands were subjected to DNA sequencing for investigation of any gene variation. Results: In this study, no mutation was found in the two exons of TMC1 gene. It was concluded from these results that mutations of the TMC1 gene’s special exons 7 and 13 have a low contribution in patients and are not great of clinical importance in these Iranian provinces. Conclusions: More studies are needed to investigate the relationship between other parts of this gene with hearing loss in different populations through the country. More research could clarify the role of this gene and its relation with deafness and provide essential information for the prevention and management of auditory disorders caused by genetic factors in the Iranian population. PMID:27247785

  14. A Brassica Exon Array for Whole-Transcript Gene Expression Profiling

    PubMed Central

    Love, Christopher G.; Graham, Neil S.; Ó Lochlainn, Seosamh; Bowen, Helen C.; May, Sean T.; White, Philip J.

    2010-01-01

    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. PMID:20862292

  15. Population Genetics of Duplicated Alternatively Spliced Exons of the Dscam Gene in Daphnia and Drosophila

    PubMed Central

    Brites, Daniela; Encinas-Viso, Francisco; Ebert, Dieter; Du Pasquier, Louis; Haag, Christoph R.

    2011-01-01

    In insects and crustaceans, the Down syndrome cell adhesion molecule (Dscam) occurs in many different isoforms. These are produced by mutually exclusive alternative splicing of dozens of tandem duplicated exons coding for parts or whole immunoglobulin (Ig) domains of the Dscam protein. This diversity plays a role in the development of the nervous system and also in the immune system. Structural analysis of the protein suggested candidate epitopes where binding to pathogens could occur. These epitopes are coded by regions of the duplicated exons and are therefore diverse within individuals. Here we apply molecular population genetics and molecular evolution analyses using Daphnia magna and several Drosophila species to investigate the potential role of natural selection in the divergence between orthologs of these duplicated exons among species, as well as between paralogous exons within species. We found no evidence for a role of positive selection in the divergence of these paralogous exons. However, the power of this test was low, and the fact that no signs of gene conversion between paralogous exons were found suggests that paralog diversity may nonetheless be maintained by selection. The analysis of orthologous exons in Drosophila and in Daphnia revealed an excess of non-synonymous polymorphisms in the epitopes putatively involved in pathogen binding. This may be a sign of balancing selection. Indeed, in Dr. melanogaster the same derived non-synonymous alleles segregate in several populations around the world. Yet other hallmarks of balancing selection were not found. Hence, we cannot rule out that the excess of non-synonymous polymorphisms is caused by segregating slightly deleterious alleles, thus potentially indicating reduced selective constraints in the putative pathogen binding epitopes of Dscam. PMID:22174757

  16. Widespread exon skipping triggers degradation by nuclear RNA surveillance in fission yeast

    PubMed Central

    Bitton, Danny A.; Atkinson, Sophie R.; Rallis, Charalampos; Smith, Graeme C.; Ellis, David A.; Chen, Yuan Y.C.; Malecki, Michal; Codlin, Sandra; Lemay, Jean-François; Cotobal, Cristina; Bachand, François; Marguerat, Samuel; Mata, Juan; Bähler, Jürg

    2015-01-01

    Exon skipping is considered a principal mechanism by which eukaryotic cells expand their transcriptome and proteome repertoires, creating different splice variants with distinct cellular functions. Here we analyze RNA-seq data from 116 transcriptomes in fission yeast (Schizosaccharomyces pombe), covering multiple physiological conditions as well as transcriptional and RNA processing mutants. We applied brute-force algorithms to detect all possible exon-skipping events, which were widespread but rare compared to normal splicing events. Exon-skipping events increased in cells deficient for the nuclear exosome or the 5′-3′ exonuclease Dhp1, and also at late stages of meiotic differentiation when nuclear-exosome transcripts decreased. The pervasive exon-skipping transcripts were stochastic, did not increase in specific physiological conditions, and were mostly present at less than one copy per cell, even in the absence of nuclear RNA surveillance and during late meiosis. These exon-skipping transcripts are therefore unlikely to be functional and may reflect splicing errors that are actively removed by nuclear RNA surveillance. The average splicing rate by exon skipping was ∼0.24% in wild type and ∼1.75% in nuclear exonuclease mutants. We also detected approximately 250 circular RNAs derived from single or multiple exons. These circular RNAs were rare and stochastic, although a few became stabilized during quiescence and in splicing mutants. Using an exhaustive search algorithm, we also uncovered thousands of previously unknown splice sites, indicating pervasive splicing; yet most of these splicing variants were cryptic and increased in nuclear degradation mutants. This study highlights widespread but low frequency alternative or aberrant splicing events that are targeted by nuclear RNA surveillance. PMID:25883323

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

    PubMed

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    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. PMID:27213357

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

    PubMed Central

    Xie, Guangrong; Yang, Weizhen; Chen, Jing; Li, Miaomiao; Jiang, Nan; Zhao, Baixue; Chen, Si; Wang, Min; Chen, Jianhua

    2016-01-01

    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. PMID:27213357

  19. Therapeutic NOTCH3 cysteine correction in CADASIL using exon skipping: in vitro proof of concept.

    PubMed

    Rutten, Julie W; Dauwerse, Hans G; Peters, Dorien J M; Goldfarb, Andrew; Venselaar, Hanka; Haffner, Christof; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke M; Lesnik Oberstein, Saskia A J

    2016-04-01

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, or CADASIL, is a hereditary cerebral small vessel disease caused by characteristic cysteine altering missense mutations in theNOTCH3gene.NOTCH3mutations in CADASIL result in an uneven number of cysteine residues in one of the 34 epidermal growth factor like-repeat (EGFr) domains of the NOTCH3 protein. The consequence of an unpaired cysteine residue in an EGFr domain is an increased multimerization tendency of mutant NOTCH3, leading to toxic accumulation of the protein in the (cerebro)vasculature, and ultimately reduced cerebral blood flow, recurrent stroke and vascular dementia. There is no therapy to delay or alleviate symptoms in CADASIL. We hypothesized that exclusion of the mutant EGFr domain from NOTCH3 would abolish the detrimental effect of the unpaired cysteine and thus prevent toxic NOTCH3 accumulation and the negative cascade of events leading to CADASIL. To accomplish this NOTCH3 cysteine correction by EGFr domain exclusion, we used pre-mRNA antisense-mediated skipping of specificNOTCH3exons. Selection of these exons was achieved usingin silicostudies and based on the criterion that skipping of a particular exon or exon pair would modulate the protein in such a way that the mutant EGFr domain is eliminated, without otherwise corrupting NOTCH3 structure and function. Remarkably, we found that this strategy closely mimics evolutionary events, where the elimination and fusion of NOTCH EGFr domains led to the generation of four functional NOTCH homologues. We modelled a selection of exon skip strategies using cDNA constructs and show that the skip proteins retain normal protein processing, can bind ligand and be activated by ligand. We then determined the technical feasibility of targetedNOTCH3exon skipping, by designing antisense oligonucleotides targeting exons 2-3, 4-5 and 6, which together harbour the majority of distinct CADASIL-causing mutations. Transfection of

  20. Control of human PLP1 expression through transcriptional regulatory elements and alternatively spliced exons in intron 1.

    PubMed

    Hamdan, Hamdan; Kockara, Neriman T; Jolly, Lee Ann; Haun, Shirley; Wight, Patricia A

    2015-01-01

    Although the myelin proteolipid protein gene (PLP1) encodes the most abundant protein in central nervous system (CNS) myelin, not much is known about the mechanisms that govern expression of the human gene (hPLP1). Much more is known about the processes that regulate Plp1 gene expression in rodents. From studies with Plp1-lacZ transgenic mice, it was determined that the first intron of mouse Plp1 (mPlp1) is required to attain high levels of expression in brain, concurrent with the active myelination period. Other studies have suggested that within mPlp1 intron 1 (>8 kb) lie several regions with enhancer-like activity. To test whether these sequences (and possibly others) in hPLP1 intron 1 are functional, deletion-transfection analysis was performed with hPLP1-lacZ constructs that contain various portions of the intron, or lack it altogether. Results presented here demonstrate the importance of hPLP1 intron 1 in achieving maximal levels of expression in the immortalized oligodendroglial cell line, Oli-neu. Deletion analysis indicates that the intron contains multiple positive regulatory elements which are active in Oli-neu cells. Some of these elements appear to be functionally conserved between human and mouse, while others are not. Furthermore, our studies demonstrate that multiple splice variants can be formed due to inclusion of extra (supplementary) exons from what is classically thought of as hPLP1 intron 1. Thus, splicing of these novel exons (which are not recognized as such in mPlp1 due to lack of conserved splice sites) must utilize factors common to both human and mouse since Oli-neu cells are of mouse origin. PMID:25694552

  1. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct N-termini.

    PubMed

    Parra, Marilyn K; Gee, Sherry L; Koury, Mark J; Mohandas, Narla; Conboy, John G

    2003-05-15

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Identified far upstream of exon 2 in both mouse and human genomes were 3 mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C; all 3 are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80-kDa 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135-kDa isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated up-regulation of 80-kDa 4.1R during terminal erythroid differentiation. Together, these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events. PMID:12522012

  2. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    SciTech Connect

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H.

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  3. Alternative 5' exons and differential splicing regulate expression of protein 4.1R isoforms with distinct n-termini

    SciTech Connect

    Parra, Marilyn K.; Gee, Sherry L.; Koury, Mark J.; Mohandas, Narla; Conboy, John G.

    2003-03-25

    Among the alternative pre-mRNA splicing events that characterize protein 4.1R gene expression, one involving exon 2' plays a critical role in regulating translation initiation and N-terminal protein structure. Exon 2' encompasses translation initiation site AUG1 and is located between alternative splice acceptor sites at the 5' end of exon 2; its inclusion or exclusion from mature 4.1R mRNA regulates expression of longer or shorter isoforms of 4.1R protein, respectively. The current study reports unexpected complexity in the 5' region of the 4.1R gene that directly affects alternative splicing of exon 2'. Three mutually exclusive alternative 5' exons, designated 1A, 1B, and 1C, were identified far upstream of exon 2 in both mouse and human genomes; all three are associated with strong transcriptional promoters in the flanking genomic sequence. Importantly, exons 1A and 1B splice differentially with respect to exon 2', generating transcripts with different 5' ends and distinct N-terminal protein coding capacity. Exon 1A-type transcripts splice so as to exclude exon 2' and therefore utilize the downstream AUG2 for translation of 80kD 4.1R protein, whereas exon 1B transcripts include exon 2' and initiate at AUG1 to synthesize 135kD isoforms. RNA blot analyses revealed that 1A transcripts increase in abundance in late erythroblasts, consistent with the previously demonstrated upregulation of 80kD 4.1R during terminal erythroid differentiation. Together these results suggest that synthesis of structurally distinct 4.1R protein isoforms in various cell types is regulated by a novel mechanism requiring coordination between upstream transcription initiation events and downstream alternative splicing events.

  4. Aberrant splicing of HTT generates the pathogenic exon 1 protein in Huntington disease.

    PubMed

    Sathasivam, Kirupa; Neueder, Andreas; Gipson, Theresa A; Landles, Christian; Benjamin, Agnesska C; Bondulich, Marie K; Smith, Donna L; Faull, Richard L M; Roos, Raymund A C; Howland, David; Detloff, Peter J; Housman, David E; Bates, Gillian P

    2013-02-01

    Huntington disease (HD) is a devastating, late-onset, inherited neurodegenerative disorder that manifests with personality changes, movement disorders, and cognitive decline. It is caused by a CAG repeat expansion in exon 1 of the HTT gene that translates to a polyglutamine tract in the huntingtin protein (HTT). The formation of HTT fragments has been implicated as an essential step in the molecular pathogenesis of HD and several proteases that cleave HTT have been identified. However, the importance of smaller N-terminal fragments has been highlighted by their presence in HD postmortem brains and by the fact that nuclear inclusions are only detected by antibodies to the N terminus of HTT. Despite an intense research effort, the precise length of these fragments and the mechanism by which they are generated remains unknown. Here we show that CAG repeat length-dependent aberrant splicing of exon 1 HTT results in a short polyadenylated mRNA that is translated into an exon 1 HTT protein. Given that mutant exon 1 HTT proteins have consistently been shown to be highly pathogenic in HD mouse models, the aberrant splicing of HTT mRNA provides a mechanistic basis for the molecular pathogenesis of HD. RNA-targeted therapeutic strategies designed to lower the levels of HTT are under development. Many of these approaches would not prevent the production of exon 1 HTT and should be reviewed in light of our findings. PMID:23341618

  5. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity.

    PubMed

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-07-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a "splicing memory" hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

  6. Exon organization of the human FKBP-12 gene: Correlation with structural and functional protein domains

    SciTech Connect

    DiLella, A.G.; Craig, R.J. )

    1991-09-03

    FKBP-12, the major T-cell binding protein for the immunosuppressive agents FK506 and rapamycin, catalyzes the interconversion of the cis and trans rotamers of the peptidyl-prolyl amide bond of peptide and protein substrates. The function of rotamase activity in cells and the role of FKBP-12 in immunoregulation is uncertain. In this paper the authors report the cloning and characterization of the human chromosomal FKBP-12 gene and four processed FKBP-12 pseudogenes. The FKBP-12 gene is 24 kilobases in length and contains five exons. The protein-coding region of the gene is divided into four exon modules that correlate with the structural and functional domains of the protein. The novel structure of FKBP-12 resulting form the topology of the antiparallel {beta}-sheet is the topological crossing of two loops that are encoded by separate exons. Separate exons also encode the antiparallel {beta}-sheet and {alpha}-helical region that define the drug-binding pocket and enzyme activity site of FKBP-12. The exon organization of the FKBP-12 gene structure will enable inactivation of this gene by homologous recombination in cells to provide a model to study the role of FKBP-12 in immunoregulation and normal cellular processes.

  7. Circular RNA biogenesis can proceed through an exon-containing lariat precursor

    PubMed Central

    Barrett, Steven P; Wang, Peter L; Salzman, Julia

    2015-01-01

    Pervasive expression of circular RNA is a recently discovered feature of eukaryotic gene expression programs, yet its function remains largely unknown. The presumed biogenesis of these RNAs involves a non-canonical ‘backsplicing’ event. Recent studies in mammalian cell culture posit that backsplicing is facilitated by inverted repeats flanking the circularized exon(s). Although such sequence elements are common in mammals, they are rare in lower eukaryotes, making current models insufficient to describe circularization. Through systematic splice site mutagenesis and the identification of splicing intermediates, we show that circular RNA in Schizosaccharomyces pombe is generated through an exon-containing lariat precursor. Furthermore, we have performed high-throughput and comprehensive mutagenesis of a circle-forming exon, which enabled us to discover a systematic effect of exon length on RNA circularization. Our results uncover a mechanism for circular RNA biogenesis that may account for circularization in genes that lack noticeable flanking intronic secondary structure. DOI: http://dx.doi.org/10.7554/eLife.07540.001 PMID:26057830

  8. iGEMS: an integrated model for identification of alternative exon usage events

    PubMed Central

    Sood, Sanjana; Szkop, Krzysztof J.; Nakhuda, Asif; Gallagher, Iain J.; Murie, Carl; Brogan, Robert J.; Kaprio, Jaakko; Kainulainen, Heikki; Atherton, Philip J.; Kujala, Urho M.; Gustafsson, Thomas; Larsson, Ola; Timmons, James A.

    2016-01-01

    DNA microarrays and RNAseq are complementary methods for studying RNA molecules. Current computational methods to determine alternative exon usage (AEU) using such data require impractical visual inspection and still yield high false-positive rates. Integrated Gene and Exon Model of Splicing (iGEMS) adapts a gene-level residuals model with a gene size adjusted false discovery rate and exon-level analysis to circumvent these limitations. iGEMS was applied to two new DNA microarray datasets, including the high coverage Human Transcriptome Arrays 2.0 and performance was validated using RT-qPCR. First, AEU was studied in adipocytes treated with (n = 9) or without (n = 8) the anti-diabetes drug, rosiglitazone. iGEMS identified 555 genes with AEU, and robust verification by RT-qPCR (∼90%). Second, in a three-way human tissue comparison (muscle, adipose and blood, n = 41) iGEMS identified 4421 genes with at least one AEU event, with excellent RT-qPCR verification (95%, n = 22). Importantly, iGEMS identified a variety of AEU events, including 3′UTR extension, as well as exon inclusion/exclusion impacting on protein kinase and extracellular matrix domains. In conclusion, iGEMS is a robust method for identification of AEU while the variety of exon usage between human tissues is 5–10 times more prevalent than reported by the Genotype-Tissue Expression consortium using RNA sequencing. PMID:27095197

  9. Intrasplicing coordinates alternative first exons with alternative splicing in the protein 4.1R gene

    SciTech Connect

    Conboy, John G.; Parra, Marilyn K.; Tan, Jeff S.; Mohandas, Narla; Conboy, John G.

    2008-11-07

    In the protein 4.1R gene, alternative first exons splice differentially to alternative 3' splice sites far downstream in exon 2'/2 (E2'/2). We describe a novel intrasplicing mechanism by which exon 1A (E1A) splices exclusively to the distal E2'/2 acceptor via two nested splicing reactions regulated by novel properties of exon 1B (E1B). E1B behaves as an exon in the first step, using its consensus 5' donor to splice to the proximal E2'/2 acceptor. A long region of downstream intron is excised, juxtaposing E1B with E2'/2 to generate a new composite acceptor containing the E1B branchpoint/pyrimidine tract and E2 distal 3' AG-dinucleotide. Next, the upstream E1A splices over E1B to this distal acceptor, excising the remaining intron plus E1B and E2' to form mature E1A/E2 product. We mapped branch points for both intrasplicing reactions and demonstrated that mutation of the E1B 5' splice site or branchpoint abrogates intrasplicing. In the 4.1R gene, intrasplicing ultimately determines N-terminal protein structure and function. More generally, intrasplicing represents a new mechanism whereby alternative promoters can be coordinated with downstream alternative splicing.

  10. Associations between intronic non-B DNA structures and exon skipping

    PubMed Central

    Tsai, Zing Tsung-Yeh; Chu, Wen-Yi; Cheng, Jen-Hao; Tsai, Huai-Kuang

    2014-01-01

    Non-B DNA structures are abundant in the genome and are often associated with critical biological processes, including gene regulation, chromosome rearrangement and genome stabilization. In particular, G-quadruplex (G4) may affect alternative splicing based on its ability to impede the activity of RNA polymerase II. However, the specific role of non-B DNA structures in splicing regulation still awaits investigation. Here, we provide a genome-wide and cross-species investigation of the associations between five non-B DNA structures and exon skipping. Our results indicate a statistically significant correlation of each examined non-B DNA structures with exon skipping in both human and mouse. We further show that the contributions of non-B DNA structures to exon skipping are influenced by the occurring region. These correlations and contributions are also significantly different in human and mouse. Finally, we detailed the effects of G4 by showing that occurring on the template strand and the length of G-run, which is highly related to the stability of a G4 structure, are significantly correlated with exon skipping activity. We thus show that, in addition to the well-known effects of RNA and protein structure, the relative positional arrangement of intronic non-B DNA structures may also impact exon skipping. PMID:24153112

  11. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    PubMed

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients. PMID:20030230

  12. Accurate quantification of dystrophin mRNA and exon skipping levels in duchenne muscular dystrophy.

    PubMed

    Spitali, Pietro; Heemskerk, Hans; Vossen, Rolf H A M; Ferlini, Alessandra; den Dunnen, Johan T; 't Hoen, Peter A C; Aartsma-Rus, Annemieke

    2010-09-01

    Antisense oligonucleotide (AON)-mediated exon skipping aimed at restoring the reading frame is a promising therapeutic approach for Duchenne muscular dystrophy that is currently tested in clinical trials. Numerous AONs have been tested in (patient-derived) cultured muscle cells and the mdx mouse model. The main outcome to measure AON efficiency is usually the exon-skipping percentage, though different groups use different methods to assess these percentages. Here, we compare a series of techniques to quantify exon skipping levels in AON-treated mdx mouse muscle. We compared densitometry of RT-PCR products on ethidium bromide-stained agarose gels, primary and nested RT-PCR followed by bioanalyzer analysis and melting curve analysis. The digital array system (Fluidigm) allows absolute quantification of skipped vs non-skipped transcripts and was used as a reference. Digital array results show that 1 ng of mdx gastrocnemius muscle-derived mRNA contains approximately 1100 dystrophin transcripts and that 665 transcripts are sufficient to determine exon-skipping levels. Quantification using bioanalyzer or densitometric analysis of primary PCR products resulted in values close to those obtained with digital array. The use of the same technique allows comparison between different groups working on exon skipping in the mdx mouse model. PMID:20458276

  13. rMAPS: RNA map analysis and plotting server for alternative exon regulation

    PubMed Central

    Park, Juw Won; Jung, Sungbo; Rouchka, Eric C.; Tseng, Yu-Ting; Xing, Yi

    2016-01-01

    RNA-binding proteins (RBPs) play a critical role in the regulation of alternative splicing (AS), a prevalent mechanism for generating transcriptomic and proteomic diversity in eukaryotic cells. Studies have shown that AS can be regulated by RBPs in a binding-site-position dependent manner. Depending on where RBPs bind, splicing of an alternative exon can be enhanced or suppressed. Therefore, spatial analyses of RBP motifs and binding sites around alternative exons will help elucidate splicing regulation by RBPs. The development of high-throughput sequencing technologies has allowed transcriptome-wide analyses of AS and RBP–RNA interactions. Given a set of differentially regulated alternative exons obtained from RNA sequencing (RNA-seq) experiments, the rMAPS web server (http://rmaps.cecsresearch.org) performs motif analyses of RBPs in the vicinity of alternatively spliced exons and creates RNA maps that depict the spatial patterns of RBP motifs. Similarly, rMAPS can also perform spatial analyses of RBP–RNA binding sites identified by cross-linking immunoprecipitation sequencing (CLIP-seq) experiments. We anticipate rMAPS will be a useful tool for elucidating RBP regulation of alternative exon splicing using high-throughput sequencing data. PMID:27174931

  14. rMAPS: RNA map analysis and plotting server for alternative exon regulation.

    PubMed

    Park, Juw Won; Jung, Sungbo; Rouchka, Eric C; Tseng, Yu-Ting; Xing, Yi

    2016-07-01

    RNA-binding proteins (RBPs) play a critical role in the regulation of alternative splicing (AS), a prevalent mechanism for generating transcriptomic and proteomic diversity in eukaryotic cells. Studies have shown that AS can be regulated by RBPs in a binding-site-position dependent manner. Depending on where RBPs bind, splicing of an alternative exon can be enhanced or suppressed. Therefore, spatial analyses of RBP motifs and binding sites around alternative exons will help elucidate splicing regulation by RBPs. The development of high-throughput sequencing technologies has allowed transcriptome-wide analyses of AS and RBP-RNA interactions. Given a set of differentially regulated alternative exons obtained from RNA sequencing (RNA-seq) experiments, the rMAPS web server (http://rmaps.cecsresearch.org) performs motif analyses of RBPs in the vicinity of alternatively spliced exons and creates RNA maps that depict the spatial patterns of RBP motifs. Similarly, rMAPS can also perform spatial analyses of RBP-RNA binding sites identified by cross-linking immunoprecipitation sequencing (CLIP-seq) experiments. We anticipate rMAPS will be a useful tool for elucidating RBP regulation of alternative exon splicing using high-throughput sequencing data. PMID:27174931

  15. iGEMS: an integrated model for identification of alternative exon usage events.

    PubMed

    Sood, Sanjana; Szkop, Krzysztof J; Nakhuda, Asif; Gallagher, Iain J; Murie, Carl; Brogan, Robert J; Kaprio, Jaakko; Kainulainen, Heikki; Atherton, Philip J; Kujala, Urho M; Gustafsson, Thomas; Larsson, Ola; Timmons, James A

    2016-06-20

    DNA microarrays and RNAseq are complementary methods for studying RNA molecules. Current computational methods to determine alternative exon usage (AEU) using such data require impractical visual inspection and still yield high false-positive rates. Integrated Gene and Exon Model of Splicing (iGEMS) adapts a gene-level residuals model with a gene size adjusted false discovery rate and exon-level analysis to circumvent these limitations. iGEMS was applied to two new DNA microarray datasets, including the high coverage Human Transcriptome Arrays 2.0 and performance was validated using RT-qPCR. First, AEU was studied in adipocytes treated with (n = 9) or without (n = 8) the anti-diabetes drug, rosiglitazone. iGEMS identified 555 genes with AEU, and robust verification by RT-qPCR (∼90%). Second, in a three-way human tissue comparison (muscle, adipose and blood, n = 41) iGEMS identified 4421 genes with at least one AEU event, with excellent RT-qPCR verification (95%, n = 22). Importantly, iGEMS identified a variety of AEU events, including 3'UTR extension, as well as exon inclusion/exclusion impacting on protein kinase and extracellular matrix domains. In conclusion, iGEMS is a robust method for identification of AEU while the variety of exon usage between human tissues is 5-10 times more prevalent than reported by the Genotype-Tissue Expression consortium using RNA sequencing. PMID:27095197

  16. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity

    PubMed Central

    Marquez, Yamile; Höpfler, Markus; Ayatollahi, Zahra; Barta, Andrea; Kalyna, Maria

    2015-01-01

    Alternative splicing (AS) diversifies transcriptomes and proteomes and is widely recognized as a key mechanism for regulating gene expression. Previously, in an analysis of intron retention events in Arabidopsis, we found unusual AS events inside annotated protein-coding exons. Here, we also identify such AS events in human and use these two sets to analyse their features, regulation, functional impact, and evolutionary origin. As these events involve introns with features of both introns and protein-coding exons, we name them exitrons (exonic introns). Though exitrons were detected as a subset of retained introns, they are clearly distinguishable, and their splicing results in transcripts with different fates. About half of the 1002 Arabidopsis and 923 human exitrons have sizes of multiples of 3 nucleotides (nt). Splicing of these exitrons results in internally deleted proteins and affects protein domains, disordered regions, and various post-translational modification sites, thus broadly impacting protein function. Exitron splicing is regulated across tissues, in response to stress and in carcinogenesis. Intriguingly, annotated intronless genes can be also alternatively spliced via exitron usage. We demonstrate that at least some exitrons originate from ancestral coding exons. Based on our findings, we propose a “splicing memory” hypothesis whereby upon intron loss imprints of former exon borders defined by vestigial splicing regulatory elements could drive the evolution of exitron splicing. Altogether, our studies show that exitron splicing is a conserved strategy for increasing proteome plasticity in plants and animals, complementing the repertoire of AS events. PMID:25934563

  17. Antisense oligonucleotide–mediated MDM4 exon 6 skipping impairs tumor growth

    PubMed Central

    Dewaele, Michael; Tabaglio, Tommaso; Willekens, Karen; Bezzi, Marco; Teo, Shun Xie; Low, Diana H.P.; Koh, Cheryl M.; Rambow, Florian; Fiers, Mark; Rogiers, Aljosja; Radaelli, Enrico; Al-Haddawi, Muthafar; Tan, Soo Yong; Hermans, Els; Amant, Frederic; Yan, Hualong; Lakshmanan, Manikandan; Koumar, Ratnacaram Chandrahas; Lim, Soon Thye; Derheimer, Frederick A.; Campbell, Robert M.; Bonday, Zahid; Tergaonkar, Vinay; Shackleton, Mark; Blattner, Christine; Marine, Jean-Christophe; Guccione, Ernesto

    2015-01-01

    MDM4 is a promising target for cancer therapy, as it is undetectable in most normal adult tissues but often upregulated in cancer cells to dampen p53 tumor-suppressor function. The mechanisms that underlie MDM4 upregulation in cancer cells are largely unknown. Here, we have shown that this key oncogenic event mainly depends on a specific alternative splicing switch. We determined that while a nonsense-mediated, decay-targeted isoform of MDM4 (MDM4-S) is produced in normal adult tissues as a result of exon 6 skipping, enhanced exon 6 inclusion leads to expression of full-length MDM4 in a large number of human cancers. Although this alternative splicing event is likely regulated by multiple splicing factors, we identified the SRSF3 oncoprotein as a key enhancer of exon 6 inclusion. In multiple human melanoma cell lines and in melanoma patient–derived xenograft (PDX) mouse models, antisense oligonucleotide–mediated (ASO-mediated) skipping of exon 6 decreased MDM4 abundance, inhibited melanoma growth, and enhanced sensitivity to MAPK-targeting therapeutics. Additionally, ASO-based MDM4 targeting reduced diffuse large B cell lymphoma PDX growth. As full-length MDM4 is enhanced in multiple human tumors, our data indicate that this strategy is applicable to a wide range of tumor types. We conclude that enhanced MDM4 exon 6 inclusion is a common oncogenic event and has potential as a clinically compatible therapeutic target. PMID:26595814

  18. Frameshift deletions of exons 3-7 and revertant fibers in Duchenne muscular dystrophy: mechanisms of dystrophin production.

    PubMed Central

    Winnard, A V; Mendell, J R; Prior, T W; Florence, J; Burghes, A H

    1995-01-01

    Duchenne muscular dystrophy (DMD) patients with mutations that disrupt the translational reading frame produce little or no dystrophin. Two exceptions are the deletion of exons 3-7 and the occurrence of rare dystrophin-positive fibers (revertant fibers) in muscle of DMD patients. Antibodies directed against the amino-terminus and the 5' end of exon 8 did not detect dystrophin in muscle from patients who have a deletion of exons 3-7. However, in all cases, dystrophin was detected with an antibody directed against the 3' end of exon 8. The most likely method of dystrophin production in these cases is initiation at a new start codon in exon 8. We also studied two patients who have revertant fibers: one had an inherited duplication of exons 5-7, which, on immunostaining, showed two types of revertant fibers; and the second patient had a 2-bp nonsense mutation in exon 51, which creates a cryptic splice site. An in-frame mRNA that uses this splice site in exon 51 was detected. Immunostaining demonstrated the presence of the 3' end of exon 51, which is in agreement with the use of this mRNA in revertant fibers. The most likely method of dystrophin production in these fibers is a second mutation that restores the reading frame. Images Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7825572

  19. Aberrant splicing and truncated-protein expression due to a newly identified XPA gene mutation.

    PubMed

    Sato, M; Nishigori, C; Yagi, T; Takebe, H

    1996-02-15

    A group A xeroderma pigmentosum (XPA) patient, XP2NI, is a compound heterozygote with a newly identified G to C transversion at the last nucleotide in exon 5 in one chromosome, and with the known splicing mutation in intron 3 in another chromosome in the XPA gene. XP2NI had mild skin symptoms and the cells were slightly less sensitive to UV radiation than the cells of typical severe XPA patients who have the splicing mutation in intron 3 homozygously. Reverse transcriptase (RT)-PCR and sequencing of the PCR products revealed that the mutation in exon 5 resulted in producing three types of aberrant mRNA, lacking 7 nucleotides at the end of exon 5, lacking entire exon 5, and lacking exons 3, 4 and 5. A significant amount of a truncated type of protein was produced in XP2NI cells, and the size of the protein indicated that it should have been translated from the mRNA, lacking the 7 nucleotides and retained one of the zinc-finger domains required for the DNA repair activity. The clinical mildness of XP2NI may be due to the residual DNA repair activity of the truncated XPA protein, while no XPA protein was detected in the XPA cells with the homozygous intron 3 splicing mutation. PMID:8596539

  20. Genetic characterization of MHC class II DQB exon 2 variants in gayal (Bos frontalis)

    PubMed Central

    Sun, Yongke; Xi, Dongmei; Li, Guozhi; Hao, Tiantian; Chen, Yuhan; Yang, Yuai

    2014-01-01

    In the present study, exon 2 of major histocompatibility complex (MHC) class II DQB gene from 39 gayals (Bos frontalis) was isolated, characterized and compared with previously reported patterns for other bovidae. It was revealed by sequence analyses that there are 36 DQB exon 2 variants among 39 gayals. These variants exhibited a high degree of nucleotide and amino acid substitutions with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (d S) and non-synonymous (d N) substitution. The gayals were observed to be under strong balancing selection in the DQB exon 2 PBS (d N = 0.094, P = 0.001). It appears that this variability among gayals could confer the ability to mount immune responses to a wide variety of peptides or pathogens. PMID:26019566

  1. Exonic STK11 deletions are not a rare cause of Peutz‐Jeghers syndrome

    PubMed Central

    Hearle, N C M; Rudd, M F; Lim, W; Murday, V; Lim, A G; Phillips, R K; Lee, P W; O'Donohue, J; Morrison, P J; Norman, A; Hodgson, S V; Lucassen, A; Houlston, R S

    2006-01-01

    Background Peutz‐Jeghers syndrome (PJS) is a rare, autosomal dominant cancer predisposition syndrome characterised by oro‐facial pigmentation and hamartomatous polyposis of the gastrointestinal tract. A causal germline mutation in STK11 can be identified in 30% to 80% of PJS patients. Methods Here we report the comprehensive mutational analysis of STK11 in 38 PJS probands applying conventional PCR based mutation detection methods and the recently introduced MLPA (multiplex ligation dependent probe amplification) technique developed for the identification of exonic deletions/duplications. Results Nineteen of 38 probands (50%) had detectable point mutations or small scale deletions/insertions and six probands (16%) had genomic deletions encompassing one or more STK11 exons. Conclusions These findings demonstrate that exonic STK11 deletions are a common cause of PJS and provide a strong rationale for conducting a primary screen for such mutations in patients. PMID:16582077

  2. In Vitro Assays to Assess Exon Skipping in Duchenne Muscular Dystrophy.

    PubMed

    Boisguerin, Prisca; O'Donovan, Liz; Gait, Michael J; Lebleu, Bernard

    2015-01-01

    Cell-penetrating peptide (CPP)-mediated delivery of phosphorodiamidate morpholino oligomers (PMO) results in efficient exon skipping and has shown great promise as a potential therapy for Duchenne muscular dystrophy (DMD). However, large differences in efficiency have been observed between CPPs and in delivery to different tissues. Cellular trafficking has appeared to be an important determinant of activity. This chapter provides details of experimental procedures to monitor exon skipping efficiency and cellular trafficking of Pip6a-PMO, a recently developed and particularly efficient conjugate, in skeletal H2k cells and in primary cardiomyocytes from mdx mice. Similar procedures may be used in principle to evaluate any free or vector-associated oligonucleotide for exon skipping. PMID:26202278

  3. Immunochemical detection of proteins related to the human c-myc exon 1.

    PubMed Central

    Gazin, C; Rigolet, M; Briand, J P; Van Regenmortel, M H; Galibert, F

    1986-01-01

    Published sequence data of the human c-myc gene indicate the presence of a coding capacity for a polypeptide of 188 residues within the first exon. Using antibodies raised against five synthetic peptides corresponding to different non-over-lapping parts of this polypeptide, two proteins of 32 kd and 58 kd antigenically related to the synthetic peptides have been detected in extracts of human cells. The confidence of this detection has been reinforced by showing that epitopes corresponding to different peptides were indeed located on the same molecule and that the 58 kd protein appears to be a dimeric form of the 32 kd protein. That these proteins originate from the first exon was indicated by: hybrid-arrested translation experiments followed by immunodetection of the translation products; in vitro translation of messenger RNA derived from cloned exon 1 by SP6 polymerase transcription. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. Fig. 7. PMID:2430795

  4. POLG exon 22 skipping induced by different mechanisms in two unrelated cases of Alpers syndrome.

    PubMed

    Mousson de Camaret, Bénédicte; Chassagne, Maïté; Mayençon, Martine; Padet, Sylvie; Crehalet, Hervé; Clerc-Renaud, Pascale; Rouvet, Isabelle; Zabot, Marie-Thérèse; Rivier, François; Sarda, Pierre; des Portes, Vincent; Bozon, Dominique

    2011-01-01

    The POLG genes were sequenced in two unrelated patients presenting with Alpers syndrome. The novel c.3626_3629dupGATA and the c.3643+2T>C alleles were associated in trans with p.A467T and p.[W748S;E1143G], respectively. POLG transcripts from skin fibroblasts showed complete exon 22 skipping for patient 2, but surprisingly partial exon 22 skipping from the c.3626_3629dupGATA for patient 1. The creation of a putative exonic splicing silencer could be responsible for the splicing anomaly observed in patient 1. Both c.3643+2T>C and c.3626_3629dupGATA create a premature termination codon and a low polymerase γ activity in skin fibroblasts is responsible for the severe phenotype in these patients. PMID:20691285

  5. The stop mutation R553X in the CFTR gene results in exon skipping

    SciTech Connect

    Hull, J.; Shackleton, S.; Harris, A. )

    1994-01-15

    Stop or nonsense mutations are known to disrupt gene function in a number of different ways. The authors have studied the effects of the stop mutation R553X in exon 11 of the CFTR gene by analyzing mRNA extracted from nasal epithelial cells harvested from patients with cystic fibrosis. Four patients who were compound heterozygotes for the R553X mutation were studied. Ten non-CF control subjects were also studied. In all four patients, full-length CFTR mRNA was identified, but only a very small proportion of this was derived from the R553X allele. A smaller transcript, lacking exon 11, was also seen in the R553X patients but not in the controls. Most of this transcript was derived from the R553X allele. These results suggest that the R553X mutation results in skipping of the exon in which it is located. 14 refs., 3 figs.

  6. Identification of Promotor and Exonic Variations, and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia

    PubMed Central

    Kar, Anjana; Munjal, Sachin Dev; Sarangi, Aditya N.; Dalal, Ashwin; Aggarwal, Rakesh

    2015-01-01

    Background Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)7TAA nucleotide sequence instead of the usual A(TA)6TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India. Methods DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1. Results Of 71 subjects with UH (68 male; median age [range], 26 [16–63] years; serum bilirubin 56 [26–219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)7TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein. Conclusion Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)7TAA

  7. Isolation of candidate genes from the Batten Disease (JNCL) region using exon amplification

    SciTech Connect

    Lerner, T.J.; Haines, J.L.; Buckler, A. |

    1994-09-01

    Batten Disease (juvenile neuronal cercoid lipofuscinosis; JNCL) is the most common neurodegenerative disorder of childhood. Clinically, this autosomal recessive disorder is characterized by loss of vision, seizures, and progressive encephalopathy. The JNCL gene CLN3 has been genetically mapped to a 2 cM region in 16p12.1-11.2 and shows significant allelic association with alleles at five marker loci, D16S288, D16S272, D16S299, D16S298, and SPN, within this interval. Extended haplotype analysis strongly suggests that CLN3 must lie in the vicinity of D16S299/D16S298. We have used these markers as STSs to isolate YACs from the CEPH and Los Alamos flow-sorted chromosome 16 YAC libraries. These YACs, in turn, have been used to generate cosmid contigs by hybridization of inter-Alu PCR products to the gridded Los Alamos chr. 16 cosmid library and by direct sub-cloning. In order to identify genes originating from this region we have used the technique of exon amplification. This procedure produces trapped exon clones, which can represent single exons or multiple exons spliced together and is an efficient method for obtaining probes for physical mapping and for screening cDNA libraries. In all experiments, we have analyzed cosmid DNA using a modification of the original exon amplification procedure that increases both the efficiency and sensitivity of the approach. We have used the resulting exon probes to screen retina and brain cDNA libraries for candidate JNCL genes. Our preliminary studies indicate that the Batten candidate region is gene-rich.

  8. Computational analysis reveals a correlation of exon-skipping events with splicing, transcription and epigenetic factors.

    PubMed

    Ye, Zhenqing; Chen, Zhong; Lan, Xun; Hara, Stephen; Sunkel, Benjamin; Huang, Tim H-M; Elnitski, Laura; Wang, Qianben; Jin, Victor X

    2014-03-01

    Alternative splicing (AS), in higher eukaryotes, is one of the mechanisms of post-transcriptional regulation that generate multiple transcripts from the same gene. One particular mode of AS is the skipping event where an exon may be alternatively excluded or constitutively included in the resulting mature mRNA. Both transcript isoforms from this skipping event site, i.e. in which the exon is either included (inclusion isoform) or excluded (skipping isoform), are typically present in one cell, and maintain a subtle balance that is vital to cellular function and dynamics. However, how the prevailing conditions dictate which isoform is expressed and what biological factors might influence the regulation of this process remain areas requiring further exploration. In this study, we have developed a novel computational method, graph-based exon-skipping scanner (GESS), for de novo detection of skipping event sites from raw RNA-seq reads without prior knowledge of gene annotations, as well as for determining the dominant isoform generated from such sites. We have applied our method to publicly available RNA-seq data in GM12878 and K562 cells from the ENCODE consortium and experimentally validated several skipping site predictions by RT-PCR. Furthermore, we integrated other sequencing-based genomic data to investigate the impact of splicing activities, transcription factors (TFs) and epigenetic histone modifications on splicing outcomes. Our computational analysis found that splice sites within the skipping-isoform-dominated group (SIDG) tended to exhibit weaker MaxEntScan-calculated splice site strength around middle, 'skipping', exons compared to those in the inclusion-isoform-dominated group (IIDG). We further showed the positional preference pattern of splicing factors, characterized by enrichment in the intronic splice sites immediately bordering middle exons. Finally, our analysis suggested that different epigenetic factors may introduce a variable obstacle in the

  9. Exon-Skipping Antisense Oligonucleotides to Correct Missplicing in Neurogenetic Diseases

    PubMed Central

    Siva, Kavitha; Covello, Giuseppina

    2014-01-01

    Alternative splicing is an important regulator of the transcriptome. However, mutations may cause alteration of splicing patterns, which in turn leads to disease. During the past 10 years, exon skipping has been looked upon as a powerful tool for correction of missplicing in disease and progress has been made towards clinical trials. In this review, we discuss the use of antisense oligonucleotides to correct splicing defects through exon skipping, with a special focus on diseases affecting the nervous system, and the latest stage achieved in its progress. PMID:24506781

  10. Evidence for a previously unidentified upstream exon in the human oestrogen receptor gene.

    PubMed

    Keaveney, M; Klug, J; Dawson, M T; Nestor, P V; Neilan, J G; Forde, R C; Gannon, F

    1991-02-01

    The presence of a previously unidentified exon upstream of the originally described human oestrogen receptor (hOR) gene is demonstrated. This is shown to be spliced to the 5' untranslated region of the previously designated exon I. The resulting genomic structure of the human gene is thus in agreement with the structure of the mouse OR gene and highlights the conservation of an 18 amino acid upstream open-reading frame formed from the above splicing event. Taken in conjunction with previous publications this would suggest that the hOR gene is a complex transcriptional unit that contains two promoters. PMID:2015052

  11. Determining exon connectivity in complex mRNAs by nanopore sequencing.

    PubMed

    Bolisetty, Mohan T; Rajadinakaran, Gopinath; Graveley, Brenton R

    2015-01-01

    Short-read high-throughput RNA sequencing, though powerful, is limited in its ability to directly measure exon connectivity in mRNAs that contain multiple alternative exons located farther apart than the maximum read length. Here, we use the Oxford Nanopore MinION sequencer to identify 7,899 'full-length' isoforms expressed from four Drosophila genes, Dscam1, MRP, Mhc, and Rdl. These results demonstrate that nanopore sequencing can be used to deconvolute individual isoforms and that it has the potential to be a powerful method for comprehensive transcriptome characterization. PMID:26420219

  12. Role of an inhibitory pyrimidine element and polypyrimidine tract binding protein in repression of a regulated alpha-tropomyosin exon.

    PubMed

    Gooding, C; Roberts, G C; Smith, C W

    1998-01-01

    Splicing of exons 2 and 3 of a-tropomyosin (TM) involves mutually exclusive selection of either exon 3, which occurs in most cells, or of exon 2 in smooth muscle (SM) cells. The SM-specific selection of exon 2 results from the inhibition of exon 3. At least two essential cis-acting elements are required for exon 3 inhibition, the upstream and downstream regulatory elements (URE and DRE). These elements are essential for repression of TM exon 3 in SM cells, and also mediate a low level of repression of exon 3 in an in vitro 5' splice site competition assay in HeLa extracts. Here, we show that the DRE consists of at least two discrete components, a short region containing a number of UGC motifs, and an essential pyrimidine-rich tract (DY). We show that the specific sequence of the DY element is important and that DY is able to bind to factors in HeLa nuclear extracts that mediate a low background level of exon 3 skipping. Deletion of a sequence within DY identified as an optimal binding site for PTB impairs (1) regulation of splicing in vivo, (2) skipping of exon 3 in an in vitro 5' splice site competition, (3) the ability of DY competitors to affect the 5' splice site competition in vitro, and (4) binding of PTB to DY. Addition of recombinant PTB to in vitro splicing reactions is able to partially reverse the effects of the DY competitor RNA. The data are consistent with a model for regulation of TM splicing that involves the participation of both tissue-specific and general inhibitory factors and in which PTB plays a role in repressing both splice sites of exon 3. PMID:9436911

  13. Exon-Specific U1s Correct SPINK5 Exon 11 Skipping Caused by a Synonymous Substitution that Affects a Bifunctional Splicing Regulatory Element.

    PubMed

    Dal Mas, Andrea; Fortugno, Paola; Donadon, Irving; Levati, Lauretta; Castiglia, Daniele; Pagani, Franco

    2015-05-01

    The c.891C>T synonymous transition in SPINK5 induces exon 11 (E11) skipping and causes Netherton syndrome (NS). Using a specific RNA-protein interaction assay followed by mass spectrometry analysis along with silencing and overexpression of splicing factors, we showed that this mutation affects an exonic bifunctional splicing regulatory element composed by two partially overlapping silencer and enhancer sequences, recognized by hnRNPA1 and Tra2β splicing factors, respectively. The C-to-T substitution concomitantly increases hnRNPA1 and weakens Tra2β-binding sites, leading to pathological E11 skipping. In hybrid minigenes, exon-specific U1 small nuclear RNAs (ExSpe U1s) that target by complementarity intronic sequences downstream of the donor splice site rescued the E11 skipping defect caused by the c.891C>T mutation. ExSpe U1 lentiviral-mediated transduction of primary NS keratinocytes from a patient bearing the mutation recovered the correct full-length SPINK5 mRNA and the corresponding functional lympho-epithelial Kazal-type related inhibitor protein in a dose-dependent manner. This study documents the reliability of a mutation-specific, ExSpe U1-based, splicing therapy for a relatively large subset of European NS patients. Usage of ExSpe U1 may represent a general approach for correction of splicing defects affecting skin disease genes. PMID:25665175

  14. Exon redefinition by a point mutation within exon 5 of the glucose-6-phosphatase gene is the major cause of glycogen storage disease type 1a in Japan

    SciTech Connect

    Kajihara, Susumu; Yamamoto, Kyosuke; Kido, Keiko

    1995-09-01

    Glycogen storage disease (GSD) type 1a (von Gierke disease) is an autosomal recessive disorder caused by a deficiency in microsomal glucose-6-phosphatase (G6Pase). We have identified a novel mutation in the G6Pase gene of a individual with GSD type 1a. The cDNA from the patient`s liver revealed a 91-nt deletion in exon 5. The genomic DNA from the patient`s white blood cells revealed no deletion or mutation at the splicing junction of intron 4 and exon 5. The 3{prime} splicing occurred 91 bp from the 5{prime} site of exon 5 (at position 732 in the coding region), causing a substitution of a single nucleotide (G to T) at position 727 in the coding region. Further confirmation of the missplicing was obtained by transient expression of allelic minigene constructs into animal cells. Another eight unrelated families of nine Japanese patients were all found to have this mutation. This mutation is a new type of splicing mutation in the G6Pase gene, and 91% of patients and carriers suffering from GSD1a in Japan are detectable with this splicing mutation. 28 refs., 5 figs., 2 tabs.

  15. Regulated splicing of the fibronectin EDA exon is essential for proper skin wound healing and normal lifespan

    PubMed Central

    Muro, Andrés F.; Chauhan, Anil K.; Gajovic, Srecko; Iaconcig, Alessandra; Porro, Fabiola; Stanta, Giorgio; Baralle, Francisco E.

    2003-01-01

    Fibronectins (FNs) are multifunctional high molecular weight glycoproteins present in the blood plasma and in the ECMs of tissues. The FN primary transcript undergoes alternative splicing in three regions generating up to 20 main different variants in humans. However, the precise role of the FN isoforms is poorly understood. One of the alternatively spliced exons is the extra domain A (EDA) or extra type III homology that is regulated spatially and temporally during development and aging. To study its in vivo function, we generated mice devoid of EDA exon-regulated splicing. Constitutive exon inclusion was obtained by optimizing the splice sites, whereas complete exclusion was obtained after in vivo CRE-loxP–mediated deletion of the exon. Homozygous mouse strains with complete exclusion or inclusion of the EDA exon were viable and developed normally, indicating that the alternative splicing at the EDA exon is not necessary during embryonic development. Conversely, mice without the EDA exon in the FN protein displayed abnormal skin wound healing, whereas mice having constitutive inclusion of the EDA exon showed a major decrease in the FN levels in all tissues. Moreover, both mutant mouse strains have a significantly shorter lifespan than the control mice, suggesting that EDA splicing regulation is necessary for efficient long-term maintenance of biological functions. PMID:12847088

  16. High frequency of JAK2 exon 12 mutations in Korean patients with polycythaemia vera: novel mutations and clinical significance.

    PubMed

    Park, Chang-Hun; Lee, Ki-O; Jang, Jun-Ho; Jung, Chul Won; Kim, Jong-Won; Kim, Sun-Hee; Kim, Hee-Jin

    2016-08-01

    Gain-of-function mutations in JAK2 are the molecular hallmarks of polycythaemia vera (PV), one of the myeloproliferative neoplasms. Most (∼95%) patients harbour V617F mutation in exon 15, while the rest have small insertion/deletion mutations in exon 12. We investigated JAK2 mutations in 42 Korean patients with PV. V617F was detected by sequencing and allele-specific PCR. When V617F was negative, sequencing and fragment length analyses were performed to detect exon 12 mutations. As a result, all patients had JAK2 mutations: 37 (88%) harboured V617F, and 5 (12%) had exon 12 mutations. Two patients had novel exon 12 mutations (H538_R541delinsLII and F537_K539delinsVL). Genotype-phenotype correlations demonstrated lower white blood cell and platelet counts in exon 12 mutations than V617F. The frequency of JAK2 exon 12 mutations was higher than expected in Korean patients with PV. Molecular genetic testing for JAK2 exon 12 mutations is mandatory for diagnosis and genotype-phenotype correlations in patients with erythrocytosis and suspected PV. PMID:27198504

  17. Investigation of biochemical changes of the ovine calpain 3 exon-10 polymorphism.

    PubMed

    Muto, Yukiyo; Morton, Jim; Palmer, David

    2015-12-01

    Calpain 3 (CAPN3) is a tissue specific calpain, and its mRNA is the most expressed calpain isoform in skeletal muscles. Many mutations and polymorphisms within the human CAPN3 gene have been reported and related to limb-girdle muscular dystrophy. Several reports link CAPN3 polymorphisms and meat quality. An association between three allele variants in exon-10 of ovine CAPN3 and the yield of fat trimmed meat cuts has been reported. This research investigated the biochemical significance of polymorphic variation in CAPN3. CAPN3 mRNA sequences were obtained from muscle samples collected from lambs which were homozygous for each of the three alleles. Four single base substitutions were found besides those in exon-10, but none of them, including the variations within exon-10, caused a change in amino acid sequence. The expression of CAPN3 mRNA and the amounts of CAPN3 protein were also compared among genotypes, and no significant differences were found. These results suggest that the reported association of specific allele variants within CAPN3 exon-10 to phenotype variations were not direct effects of CAPN3 polymorphisms. Interspecies analyses of the CAPN3 sequences indicated that the sequence reported here is more likely to be the correct common ovine CAPN3 sequence than the reference sequence. PMID:26363096

  18. Detection EGFR exon 19 status of lung cancer patients by DNA electrochemical biosensor.

    PubMed

    Xu, Xiong-Wei; Weng, Xiu-Hua; Wang, Chang-Lian; Lin, Wei-Wei; Liu, Ai-Lin; Chen, Wei; Lin, Xin-Hua

    2016-06-15

    Epidermal growth factor receptor (EGFR) exon 19 mutation status is a very important prediction index for tyrosine kinase inhibitors (TKIs) therapy. In this paper, we constructed a superior selective sandwich-type electrochemical biosensor to detect in-frame deletions in exon 19 of EGFR in real samples of patients with non-small cell lung carcinoma. Based on the characteristics of different hybridization efficiency in different hybridization phase conditions, different region around EGFR exon 19 deletion hotspots was selected to design DNA probes to improve biosensor performance. The results confirm that alteration of deletion location in target deliberately according to different hybridization phase is able to improve selectivity of sandwich-type DNA biosensor. Satisfactory discrimination ability can be achieved when the deletions are located in the capture probe interaction region. In order to improve efficiency of ssDNA generation from dsDNA, we introduce Lambda exonuclease (λ-exo) to sandwich-type biosensor system. EGFR exon 19 statuses of clinical real samples from lung cancer patients can be discriminated successfully by the proposed method. Our research would make the electrochemical biosensor be an excellent candidate for EGFR detection for lung cancer patients. PMID:26874108

  19. Integrated Exon Level Expression Analysis of Driver Genes Explain Their Role in Colorectal Cancer

    PubMed Central

    Aziz, Mohammad Azhar; Periyasamy, Sathish; Al Yousef, Zeyad; AlAbdulkarim, Ibrahim; Al Otaibi, Majed; Alfahed, Abdulaziz; Alasiri, Glowi

    2014-01-01

    Integrated analysis of genomic and transcriptomic level changes holds promise for a better understanding of colorectal cancer (CRC) biology. There is a pertinent need to explain the functional effect of genome level changes by integrating the information at the transcript level. Using high resolution cytogenetics array, we had earlier identified driver genes by ‘Genomic Identification of Significant Targets In Cancer (GISTIC)’ analysis of paired tumour-normal samples from colorectal cancer patients. In this study, we analyze these driver genes at three levels using exon array data – gene, exon and network. Gene level analysis revealed a small subset to experience differential expression. These results were reinforced by carrying out separate differential expression analyses (SAM and LIMMA). ATP8B1 was found to be the novel gene associated with CRC that shows changes at cytogenetic, gene and exon levels. Splice index of 29 exons corresponding to 13 genes was found to be significantly altered in tumour samples. Driver genes were used to construct regulatory networks for tumour and normal groups. There were rearrangements in transcription factor genes suggesting the presence of regulatory switching. The regulatory pattern of AHR gene was found to have the most significant alteration. Our results integrate data with focus on driver genes resulting in highly enriched novel molecules that need further studies to establish their role in CRC. PMID:25335079

  20. Mutations in exon 10 of the RET proto-oncogene in Hirschsprung`s disease

    SciTech Connect

    Attie, T.; Eng, C.; Mulligan, L.M.

    1994-09-01

    Hirschsprung`s disease (HSCR) is a frequent congenital malformation ascribed to the absence of autonomic ganglion cells in the terminal hindgut. Recently, we have identified mutations in the RET proto-oncogene in HSCR families. Mutations of the RET gene have also been reported in multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). While RET mutations in HSCR are scattered on the whole coding sequence, MEN 2A and FMTC mutations are clustered in 5 cystein codons of exons 10 and 11. Here, we report on HSCR families carrying mutations in exon 10 of the RET gene, one of them involving a cystein codon. Germ-line mutations in exon 10 of the RET gene may contribute to either an early development defect (HSCR) or inherited predisposition to cancer (MEN 2A and FMTC), probable depending on the nature and location of the mutation. These data also suggest that HSCR patients with mutations in exon 10 might subsequently prove to be at risk for MEN 2A or FMTC since several MEN 2A/HSCR associations have been reported.

  1. Prenatal stress decreases Bdnf expression and increases methylation of Bdnf exon IV in rats

    PubMed Central

    Boersma, Gretha J; Lee, Richard S; Cordner, Zachary A; Ewald, Erin R; Purcell, Ryan H; Moghadam, Alexander A; Tamashiro, Kellie L

    2014-01-01

    There is ample evidence that exposure to stress during gestation increases the risk of the offspring to develop mood disorders. Brain-derived neurotrophic factor (Bdnf) plays a critical role during neuronal development and is therefore a prime candidate to modulate neuronal signaling in adult offspring of rat dams that were stressed during gestation. In the current study, we tested the hypothesis that alterations in Bdnf expression in prenatally stressed (PNS) offspring are mediated by changes in DNA methylation in exons IV and VI of the Bdnf gene. We observed decreased Bdnf expression in the amygdala and hippocampus of prenatally stressed rats both at weaning and in adulthood. This decrease in Bdnf expression was accompanied by increased DNA methylation in Bdnf exon IV in the amygdala and hippocampus, suggesting that PNS-induced reduction in Bdnf expression may, at least in part, be mediated by increased DNA methylation of Bdnf exon IV. Expression of DNA methyltransferases (Dnmt) 1 and 3a was increased in PNS rats in the amygdala and hippocampus. Our data suggest that PNS induces decreases in Bdnf expression that may at least in part be mediated by increased DNA methylation of Bdnf exon IV. PMID:24365909

  2. Disperse—a software system for design of selector probes for exon resequencing applications

    PubMed Central

    Stenberg, J.; Zhang, M.; Ji, H.

    2009-01-01

    Summary:Selector probes enable the amplification of many selected regions of the genome in multiplex. Disperse is a software pipeline that automates the procedure of designing selector probes for exon resequencing applications. Availability:Software and documentation is available at http://bioinformatics.org/disperse Contact: genomics_ji@stanford.edu PMID:19158162

  3. The monogenetic kinetoplastid protozoan, Crithidia fasciculata, contains a transcriptionally active, multicopy mini-exon sequence.

    PubMed Central

    Muhich, M L; Hughes, D E; Simpson, A M; Simpson, L

    1987-01-01

    A repeated sequence from the Crithidia fasciculata nuclear genome has been isolated which is homologous to the mini-exon genes of other kinetoplastid protozoa. Sequence analysis of the 417 bp monomeric unit confirmed the presence of a 35 nt sequence within the repeat that is 77% homologous with the Trypanosoma brucei 35-mer mini-exon or spliced leader sequence. The repeat is present at approximately 250 copies per cell and is organized into one, or a few, large head to tail tandem clusters predominantly on a single chromosome. The mini-exon repeat unit hybridizes to a major 84 nt and a minor 87 nt poly (A)- steady state transcript, the first 35 nts of which comprise the mini-exon sequence found at the 5' end of mRNAs in several other kinetoplastid species. The 3'-termini of the transcripts map to positions on the DNA sense strand directly preceeding a stretch of 8 thymidine residues. Crithidia represents the most primitive kinetoplastid species which apparently possesses a discontinuous type of mRNA processing, implying that this represents a conserved feature in possibly all genera of kinetoplastid protozoa. Images PMID:3562248

  4. Screening two mutations in the dysferlin gene by exon capture and sequence analysis: A case report

    PubMed Central

    WANG, XUEYAN; YANG, YUN; ZHOU, RONG

    2016-01-01

    A patient with progressive muscular atrophy was assessed for the disease-associated genes by next-generation sequencing technology and exon trap and sequence analysis. The results of the investigation identified 399 genes, covering all exons in addition to 10 bp on either side, which are specific to 659 types of neuromuscular disorders, including hypotypes. Exon capture and sequence analysis revealed that the patient possessed two splice site mutations in the dysferlin (DYSF) gene, c.144+1G>A and c.342+1G>T, and the presence of the mutations was confirmed by Sanger sequencing. The patient's mother and sister were also assessed and confirmed to have mutations within the DYSF gene, the mother with c.342+1G>T and the sister with c.144+1G>A. The two splice site mutations in the DYSF gene, c.144+1G>A and c.342+1G>T, have not previously been reported. Therefore, exon capture and sequence analysis is able to rapidly and efficiently screen for genetic alterations in neuromuscular disorders.

  5. A novel first exon directs hormone-sensitive transcription of the pig prolactin receptor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Endocrine, paracrine, and autocrine prolactin (PRL) acts through its receptor (PRLR) to confer a wide range of biological functions, including its established role during lactation.We have identified a novel first exon of the porcine PRLR that gives rise to three different mRNA transcripts. Transcri...

  6. Polypurine sequences within a downstream exon function as a splicing enhancer

    SciTech Connect

    Tanaka, Kenji; Watakabe, Akiya; Shimura, Yoshiro

    1994-02-01

    We have previously shown that a purine-rich sequence located within exon M2 of the mouse immunoglobulin {mu} gene functions as a splicing enhancer, as judged by its ability to stimulate splicing of a distant upstream intron. This sequence element has been designated ERS (exon recognition sequence). In this study, we investigated the stimulatory effects of various ERS-like sequences, using the in vitro splicing system with HeLa cell nuclear extracts. Here, we show that purine-rich sequences of several natural exons that have previously been shown to be required for splicing function as a splicing enhancer like the ERS of the immunoglobulin {mu} gene. Moreover, even synthetic polypurine sequences had stimulatory effects on the upstream splicing. Evaluation of the data obtained from the analyses of both natural and synthetic purine-rich sequences shows that (i) alternating purine sequences can stimulate splicing, while poly(A) or poly(G) sequences cannot, and (ii) the presence of U residues within the polypurine sequence greatly reduces the level of stimulation. Competition experiments strongly suggest that the stimulatory effects of various purine-rich sequences are mediated by the same trans-acting factor(s). We conclude from these results that the purine-rich sequences that we examined in this study also represent examples of ERS. Thus, ERS is considered a general splicing element that is present in various exons and plays an important role in splice site selection. 50 refs., 7 figs., 2 tabs.

  7. Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer.

    PubMed

    Sahoo, Bankanidhi; Arduini, Irene; Drombosky, Kenneth W; Kodali, Ravindra; Sanders, Laurie H; Greenamyre, J Timothy; Wetzel, Ronald

    2016-01-01

    Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington's disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6-9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile

  8. Cloning of Caenorhabditis U2AF65: an alternatively spliced RNA containing a novel exon.

    PubMed Central

    Zorio, D A; Lea, K; Blumenthal, T

    1997-01-01

    The U2 small nuclear ribonucleoprotein particle (snRNP) auxiliary factor, U2AF, is an essential splicing factor required for recognition of the polypyrimidine tract and subsequent U2 snRNP assembly at the branch point. Because Caenorhabditis elegans introns lack both polypyrimidine tract and branch point consensus sequences but have a very highly conserved UUUUCAG/R consensus at their 3' splice sites, we hypothesized that U2AF might serve to recognize this sequence and thus promote intron recognition in C. elegans. Here we report the cloning of the gene for the large subunit of U2AF, uaf-1. Three classes of cDNA were identified. In the most abundant class the open reading frame is similar to that for the U2AF65 from mammals and flies. The remaining two classes result from an alternative splicing event in which an exon containing an in-frame stop codon is inserted near the beginning of the second RNA recognition motif. However, this alternative mRNA is apparently not translated. Interestingly, the inserted exon contains 10 matches to the 3' splice site consensus. To determine whether this feature is conserved, we sequenced uaf-1 from the related nematode Caenorhabditis briggsae. It is composed of six exons, including an alternatively spliced third exon interrupting the gene at the same location as in C. elegans. uaf-1 is contained in an operon with the rab-18 gene in both species. Although the alternative exons from the two species are not highly conserved and would not encode related polypeptides, the C. briggsae alternative exon has 18 matches to the 3' splice site consensus. We hypothesize that the array of 3' splice site-like sequences in the pre-mRNA and alternatively spliced exon may have a regulatory role. The alternatively spliced RNA accumulates at high levels following starvation, suggesting that this RNA may represent an adaption for reducing U2AF65 levels when pre-mRNA levels are low. PMID:9001248

  9. Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer

    PubMed Central

    Sahoo, Bankanidhi; Arduini, Irene; Drombosky, Kenneth W.; Kodali, Ravindra; Sanders, Laurie H.; Greenamyre, J. Timothy; Wetzel, Ronald

    2016-01-01

    Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington’s disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6–9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction

  10. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy.

    PubMed Central

    Nguyen, T M; Morris, G E

    1993-01-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random "libraries" of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25-60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1-41, and we now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. Images Figure 4 Figure 1 PMID:7684887

  11. Characterization of a spliced exon product of herpes simplex type-1 latency-associated transcript in productively infected cells

    SciTech Connect

    Kang, Wen; Mukerjee, Ruma; Gartner, Jared J.; Hatzigeorgiou, Artemis G.; Sandri-Goldin, Rozanne M.; Fraser, Nigel W. . E-mail: nfraser@mail.med.upenn.edu

    2006-12-20

    The latency-associated transcripts (LATs) of herpes simplex virus type-1 (HSV-1) are the only viral RNAs accumulating during latent infections in the sensory ganglia of the peripheral nervous system. The major form of LAT that accumulates in latently infected neurons is a 2 kb intron, spliced from a much less abundant 8.3 primary transcript. The spliced exon mRNA has been hard to detect. However, in this study, we have examined the spliced exon RNA in productively infected cells using ribonuclease protection (RPA), and quantitative RT-PCR (q-PCR) assays. We were able to detect the LAT exon RNA in productively infected SY5Y cells (a human neuronal cell line). The level of the LAT exon RNA was found to be approximately 5% that of the 2 kb intron RNA and thus is likely to be relatively unstable. Quantitative RT-PCR (q-PCR) assays were used to examine the LAT exon RNA and its properties. They confirmed that the LAT exon mRNA is present at a very low level in productively infected cells, compared to the levels of other viral transcripts. Furthermore, experiments showed that the LAT exon mRNA is expressed as a true late gene, and appears to be polyadenylated. In SY5Y cells, in contrast to most late viral transcripts, the LAT exon RNA was found to be mainly nuclear localized during the late stage of a productive infection. Interestingly, more LAT exon RNA was found in the cytoplasm in differentiated compared to undifferentiated SY5Y cells, suggesting the nucleocytoplasmic distribution of the LAT exon RNA and its related function may be influenced by the differentiation state of cells.

  12. Use of epitope libraries to identify exon-specific monoclonal antibodies for characterization of altered dystrophins in muscular dystrophy

    SciTech Connect

    Nguyen thi Man; Morris, G.E. )

    1993-06-01

    The majority of mutations in Xp21-linked muscular dystrophy (MD) can be identified by PCR or Southern blotting, as deletions or duplications of groups of exons in the dystrophin gene, but it is not always possible to predict how much altered dystrophin, if any, will be produced. Use of exon-specific monoclonal antibodies (mAbs) on muscle biopsies from MD patients can, in principle, provide information on both the amount of altered dystrophin produced and, when dystrophin is present, the nature of the genetic deletion or point mutation. For this purpose, mAbs which recognize regions of dystrophin encoded by known exons and whose binding is unaffected by the absence of adjacent exons are required. To map mAbs to specific exons, random [open quotes]libraries[close quotes] of expressed dystrophin fragments were created by cloning DNAseI digestion fragments of a 4.3-kb dystrophin cDNA into a pTEX expression vector. The libraries were then used to locate the epitopes recognized by 48 mAbs to fragments of 25--60 amino acids within the 1,434-amino-acid dystrophin fragment used to produce the antibodies. This is sufficiently detailed to allow further refinement by using synthetic peptides and, in many cases, to identify the exon in the DMD (Duchenne MD) gene which encodes the epitope. To illustrate their use in dystrophin analysis, a Duchenne patient with a frameshift deletion of exons 42 and 43 makes a truncated dystrophin encoded by exons 1--41, and the authors now show that this can be detected in the sarcolemma by mAbs up to and including those specific for exon 41 epitopes but not by mAbs specific for exon 43 or later epitopes. 38 refs., 2 figs., 4 tabs.

  13. SNP discovery in candidate adaptive genes using exon capture in a free-ranging alpine ungulate.

    PubMed

    Roffler, Gretchen H; Amish, Stephen J; Smith, Seth; Cosart, Ted; Kardos, Marty; Schwartz, Michael K; Luikart, Gordon

    2016-09-01

    Identification of genes underlying genomic signatures of natural selection is key to understanding adaptation to local conditions. We used targeted resequencing to identify SNP markers in 5321 candidate adaptive genes associated with known immunological, metabolic and growth functions in ovids and other ungulates. We selectively targeted 8161 exons in protein-coding and nearby 5' and 3' untranslated regions of chosen candidate genes. Targeted sequences were taken from bighorn sheep (Ovis canadensis) exon capture data and directly from the domestic sheep genome (Ovis aries v. 3; oviAri3). The bighorn sheep sequences used in the Dall's sheep (Ovis dalli dalli) exon capture aligned to 2350 genes on the oviAri3 genome with an average of 2 exons each. We developed a microfluidic qPCR-based SNP chip to genotype 476 Dall's sheep from locations across their range and test for patterns of selection. Using multiple corroborating approaches (lositan and bayescan), we detected 28 SNP loci potentially under selection. We additionally identified candidate loci significantly associated with latitude, longitude, precipitation and temperature, suggesting local environmental adaptation. The three methods demonstrated consistent support for natural selection on nine genes with immune and disease-regulating functions (e.g. Ovar-DRA, APC, BATF2, MAGEB18), cell regulation signalling pathways (e.g. KRIT1, PI3K, ORRC3), and respiratory health (CYSLTR1). Characterizing adaptive allele distributions from novel genetic techniques will facilitate investigation of the influence of environmental variation on local adaptation of a northern alpine ungulate throughout its range. This research demonstrated the utility of exon capture for gene-targeted SNP discovery and subsequent SNP chip genotyping using low-quality samples in a nonmodel species. PMID:27327375

  14. Huntingtin exon 1 fibrils feature an interdigitated β-hairpin–based polyglutamine core

    PubMed Central

    Hoop, Cody L.; Lin, Hsiang-Kai; Kar, Karunakar; Magyarfalvi, Gábor; Lamley, Jonathan M.; Boatz, Jennifer C.; Mandal, Abhishek; Lewandowski, Józef R.; Wetzel, Ronald

    2016-01-01

    Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington’s disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin–containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand–based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of “intrinsic” polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms. PMID:26831073

  15. Analysis of c-KIT exon 11 mutations in canine gastrointestinal stromal tumours.

    PubMed

    Takanosu, M; Amano, S; Kagawa, Y

    2016-01-01

    The aim of this study was to determine the type and frequency of c-KIT exon 11 mutations in canine gastrointestinal stromal tumours (GISTs) and investigate the association between the c-KIT mutation status and KIT immunohistochemical staining pattern. Mutations in exon 11 of c-KIT were examined in 46 formalin-fixed paraffin-embedded canine GISTs using PCR of genomic DNA and reverse transcription-PCR (RT-PCR) of cDNA. Exon 11 c-KIT mutations were detected in 15/46 (32.6%) cases by conventional PCR and 34/46 (73.9%) cases by RT-PCR; the mutation detection rate was significantly higher for RT-PCR (P = 0.004, Fisher's exact test). Ten different mutations, including deletion, internal tandem duplication and point mutations, were identified by RT-PCR. Immunohistochemistry was performed using an anti-KIT antibody; diffuse KIT staining was detected in the tumour cell cytoplasm in 32/46 (69.6%) cases and partial or stippled cytoplasmic staining of KIT was observed in 14/46 (30.4%) cases. Neither pattern was significantly associated with c-KIT exon 11 mutation status (P = 1.000, chi-square test). These data indicate that c-KIT exon 11 mutations occur frequently in canine GISTs, similar to human GISTs; however, there is no association between c-KIT mutations and the KIT expression pattern in canine GISTs. This study suggests that RT-PCR is more sensitive than conventional PCR for the detection of c-KIT mutations in canine GISTs. PMID:26631948

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

    PubMed Central

    Madsen, Bo Eskerod; Villesen, Palle; Wiuf, Carsten

    2008-01-01

    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. PMID:18789129

  17. Lost in translation: translational interference from a recurrent mutation in exon 1 of MECP2

    PubMed Central

    Saxena, A; de Lagarde, D; Leonard, H; Williamson, S L; Vasudevan, V; Christodoulou, J; Thompson, E; MacLeod, P; Ravine, D

    2006-01-01

    Background Rett syndrome (RTT) is an X linked neuro‐developmental disorder affecting mostly girls. Mutations in the coding region of MECP2 are found in 80% of classic RTT patients. Until recently, the region encoding MECP2 was believed to comprise exons 2, 3, and 4 with the ATG start site located at the end of exon 2 (MeCP2_e2). Methods Recent reports of another mRNA transcript transcribed from exon 1 (MeCP2_e1) prompted us to screen exon 1 among RNA samples from 20 females with classic or atypical RTT. Results A previously reported 11 base pair deletion in exon 1 was detected in one subject with a milder phenotype. Although RNA expression for both protein isoforms was detected from the mutant allele, evaluation of MeCP2 protein in uncultured patient lymphocytes by immunocytochemistry revealed that MeCP2 protein production was restricted to only 74–76% of lymphocytes. X chromosome inactivation studies of genomic DNA revealed similar XCI ratios at the HUMARA locus (73:27 with HpaII and 74:26 with McrBC). We have demonstrated that translation but not transcription of the MeCP2_e2 isoform is ablated by the 11 nucleotide deletion, 103 nucleotides upstream of the e2 translation start site. Conclusions These findings reveal that nucleotides within the deleted sequence in the 5′‐UTR of the MeCP2_e2 transcript, while not required for transcription, are essential for translation. PMID:16155192

  18. NF1 Exon 22 Analysis of Individuals with the Clinical Diagnosis of Neurofibromatosis Type 1

    PubMed Central

    Muram-Zborovski, Talia M.; Vaughn, Cecily P.; Viskochil, David H.; Hanson, Heather; Mao, Rong; Stevenson, David A.

    2010-01-01

    Café-au-lait macules are frequently seen in Ras-MAPK pathway disorders and are a cardinal feature of neurofibromatosis type 1 (NF1). Most NF1 individuals develop age-related tumorigenic manifestations (e.g. neurofibromas), although individuals with a specific 3-bp deletion in exon 22 of NF1 (c.2970_2972delAAT) have an attenuated phenotype with primarily pigmentary manifestations. Previous reports identify this deletion c.2970_2972delAAT in exon 17 of NF1 using NF Consortium nomenclature. For this report, we elected to use standard NCBI nomenclature, which places this identical deletion within exon 22. SPRED1 causes Legius syndrome, which clinically overlaps with this attenuated NF1 phenotype. In an unselected cohort of 150 individuals who fulfilled NIH clinical diagnostic criteria from an NF Clinic and did not have SPRED1 mutations, we sequenced NF1 exon 22 in order to identify children and adolescents with multiple café-au-lait spots who could be projected to have lower likelihood to develop tumors. Two individuals with NF1 exon 22 mutations were identified: an 11-year-old boy with the c.2970_2972delAAT in-frame deletion and a 4-year-old boy with c.2866dupA. The father of the second patient had an attenuated form of NF1 and showed 24% germline mosaicism of the c.2866dupA mutation in whole blood. These individuals emphasize the need for mutation analysis in some individuals with the clinical diagnosis of NF1 who lack the tumorigenic or classic skeletal abnormalities of NF1. Specifically, with the identification of Legius syndrome, the need to recognize the attenuated phenotype of NF1 mosaicism and confirmation by mutation analysis is increasingly important for appropriate medical management and family counseling. PMID:20602485

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

    PubMed

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

    2015-06-01

    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

  20. Exon Organization and Novel Alternative Splicing of Ank3 in Mouse Heart

    PubMed Central

    Yamankurt, Gokay; Wu, Henry C.; McCarthy, Michael; Cunha, Shane R.

    2015-01-01

    Ankyrin-G is an adaptor protein that links membrane proteins to the underlying cytoskeletal network. Alternative splicing of the Ank3 gene gives rise to multiple ankyrin-G isoforms in numerous tissues. To date, only one ankyrin-G isoform has been characterized in heart and transcriptional regulation of the Ank3 gene is completely unknown. In this study, we describe the first comprehensive analysis of Ank3 expression in heart. Using a PCR-based screen of cardiac mRNA transcripts, we identify two new exons and 28 alternative splice variants of the Ank3 gene. We measure the relative expression of each splice variant using quantitative real-time PCR and exon-exon boundary spanning primers that specifically amplify individual Ank3 variants. Six variants are rarely expressed (<1%), while the remaining variants display similar expression patterns in three hearts. Of the five first exons in the Ank3 gene, exon 1d is only expressed in heart and skeletal muscle as it was not detected in brain, kidney, cerebellum, and lung. Immunoblot analysis reveals multiple ankyrin-G isoforms in heart, and two ankyrin-G subpopulations are detected in adult cardiomyocytes by immunofluorescence. One population co-localizes with the voltage-gated sodium channel NaV1.5 at the intercalated disc, while the other population expresses at the Z-line. Two of the rare splice variants excise a portion of the ZU5 motif, which encodes the minimal spectrin-binding domain, and these variants lack β-spectrin binding. Together, these data demonstrate that Ank3 is subject to complex splicing regulation resulting in a diverse population of ankyrin-G isoforms in heart. PMID:26024478

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

    PubMed Central

    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

  2. Sequencing of candidate genes in Dominican families implicates both rare exonic and common non-exonic variants for carotid intima-media thickness at bifurcation.

    PubMed

    Wang, Liyong; Beecham, Ashley; Dueker, Nicole; Blanton, Susan H; Rundek, Tatjana; Sacco, Ralph L

    2015-10-01

    Through linkage and tagSNP-based association studies in 100 Dominican Republic (DR) families, we previously identified ANLN and AOAH (7p14.3) as candidate genes for carotid intima-media thickness at bifurcation (bIMT). Introns, exons, and flanking regions of ANLN and AOAH were re-sequenced in 151 individuals from nine families with evidence for linkage at 7p14.3. For common variants [CV, minor allele frequency (MAF) ≥5 %], single variant-based analysis was performed. For rare variants (RV, MAF < 5 %), gene-based analysis aggregating all RVs within a gene was performed. CV analysis revealed the strongest signal at rs3815483 (P = 0.0003) in ANLN and rs60023210 (P = 0.00005) in AOAH. In ANLN, RV analysis found suggestive evidence for association with exonic RVs (P = 0.08), and in particular non-synonymous RVs (P = 0.04) but not with all RVs (P = 0.15). The variant alleles of all non-synonymous RVs segregated with the major allele of rs3815483 and were associated with lower bIMT while a novel synonymous RV segregated with the minor allele of rs3815483 and was associated with greater bIMT. Additional analysis in 561 DR individuals found suggestive evidence for association with all ANLN non-synonymous RVs (P = 0.08). In AOAH, no evidence for association with RVs was detected. Instead, conditional analysis revealed that multiple independent intronic CVs are associated with bIMT in addition to rs60023210. We demonstrate the utility of using family-based studies to evaluate the contribution of RVs. Our data suggest two modes of genetic architecture underlying the linkage and association at ANLN (multiple exonic RVs) and AOAH (multiple intronic CVs with uncharacterized functions). PMID:26319989

  3. The Drosophila erect wing gene, which is important for both neuronal and muscle development, encodes a protein which is similar to the sea urchin P3A2 DNA binding protein.

    PubMed Central

    DeSimone, S M; White, K

    1993-01-01

    The erect wing (ewg) locus of Drosophila melanogaster encodes a vital function important for the development of the nervous system and the indirect flight muscles. In order to understand the ewg function at a molecular level, cDNA clones were isolated. Sequence analysis of cDNAs revealed a single open reading frame (ORF) encoding a protein of 733 residues. The translational start for this ORF is a CTG codon. A 225-amino-acid region of this protein is 71% identical to the DNA binding region of the Strongylocentrotus purpuratus P3A2 DNA binding protein. Additionally, the ORF contains large acidic and basic domains characteristic of those in proteins involved in nuclear regulatory functions. Immunoblot analysis using polyclonal anti-EWG antisera generated against a bacterial fusion protein reveals a single, 116-kDa protein present throughout development, beginning at approximately stage 12 of embryogenesis, which is enriched in adult heads and absent from embryos carrying certain ewg alleles. Additionally, we show that EWG is localized specifically to the nuclei of virtually all embryonic neurons. Finally, a minigene consisting of an ewg cDNA under control of the hsp70 promoter can provide the ewg function in transgenic ewg mutant flies. Images PMID:8388540

  4. Delineation of the Marfan phenotype associated with mutations in exons 23-32 of the FBN1 gene

    SciTech Connect

    Putnam, E.A.; Cho, M.; Milewicz, D.M.

    1996-03-29

    Marfan syndrome is a dominantly inherited connective tissue disorder with a wide range of phenotypic severity. The condition is the result of mutations in FBN1, a large gene composed of 65 exons encoding the fibrillin-1 protein. While mutations causing classic manifestations of Marfan syndrome have been identified throughout the FBN1 gene, the six previously characterized mutations resulting in the severe, perinatal lethal form of Marfan syndrome have clustered in exons 24-32 of the gene. We screened 8 patients with either neonatal Marfan syndrome or severe cardiovascular complications of Marfan syndrome for mutations in this region of the gene. Using intron-based exon-specific primers, we amplified exons 23-32 from genomic DNAs, screened these fragments by single-stranded conformational polymorphism analysis, and sequenced indicated exons. This analysis documented mutations in exons 25-27 of the FBN1 mutations in 6 of these patients. These results, taken together with previously published FBN1 mutations in this region, further define the phenotype associated with mutations in exons 24-32 of the FBN1 gene, information important for the development of possible diagnostic tests and genetic counseling. 49 refs., 4 figs., 2 tabs.

  5. A majority of m6A residues are in the last exons, allowing the potential for 3' UTR regulation.

    PubMed

    Ke, Shengdong; Alemu, Endalkachew A; Mertens, Claudia; Gantman, Emily Conn; Fak, John J; Mele, Aldo; Haripal, Bhagwattie; Zucker-Scharff, Ilana; Moore, Michael J; Park, Christopher Y; Vågbø, Cathrine Broberg; Kusśnierczyk, Anna; Klungland, Arne; Darnell, James E; Darnell, Robert B

    2015-10-01

    We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m(6)A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3'-most (last) exons, with a very sharp rise (sixfold) within 150-400 nucleotides of the start of the last exon. Two-thirds of last exon m(6)A and >40% of all m(6)A in mRNA are present in 3' untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m(6)A sites around stop codons. Moreover, m(6)A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m(6)A density peaks early in the 3' UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m(6)A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m(6)A modification in regulating proximal alternative polyA choice. PMID:26404942

  6. A majority of m6A residues are in the last exons, allowing the potential for 3′ UTR regulation

    PubMed Central

    Ke, Shengdong; Alemu, Endalkachew A.; Mertens, Claudia; Gantman, Emily Conn; Fak, John J.; Mele, Aldo; Haripal, Bhagwattie; Zucker-Scharff, Ilana; Moore, Michael J.; Park, Christopher Y.; Vågbø, Cathrine Broberg; Kusśnierczyk, Anna; Klungland, Arne; Darnell, James E.; Darnell, Robert B.

    2015-01-01

    We adapted UV CLIP (cross-linking immunoprecipitation) to accurately locate tens of thousands of m6A residues in mammalian mRNA with single-nucleotide resolution. More than 70% of these residues are present in the 3′-most (last) exons, with a very sharp rise (sixfold) within 150–400 nucleotides of the start of the last exon. Two-thirds of last exon m6A and >40% of all m6A in mRNA are present in 3′ untranslated regions (UTRs); contrary to earlier suggestions, there is no preference for location of m6A sites around stop codons. Moreover, m6A is significantly higher in noncoding last exons than in next-to-last exons harboring stop codons. We found that m6A density peaks early in the 3′ UTR and that, among transcripts with alternative polyA (APA) usage in both the brain and the liver, brain transcripts preferentially use distal polyA sites, as reported, and also show higher proximal m6A density in the last exons. Furthermore, when we reduced m6A methylation by knocking down components of the methylase complex and then examined 661 transcripts with proximal m6A peaks in last exons, we identified a set of 111 transcripts with altered (approximately two-thirds increased proximal) APA use. Taken together, these observations suggest a role of m6A modification in regulating proximal alternative polyA choice. PMID:26404942

  7. RBFOX2 Promotes Protein 4.1R Exon 16 Selection via U1 snRNP Recruitment

    PubMed Central

    Ou, Alexander C.; Park, Jennie; Yu, Faye; Yu, Brian; Lee, Angela; Yang, Guang; Zhou, Anyu; Benz, Edward J.

    2012-01-01

    The erythroid differentiation-specific splicing switch of protein 4.1R exon 16, which encodes a spectrin/actin-binding peptide critical for erythrocyte membrane stability, is modulated by the differentiation-induced splicing factor RBFOX2. We have now characterized the mechanism by which RBFOX2 regulates exon 16 splicing through the downstream intronic element UGCAUG. Exon 16 possesses a weak 5′ splice site (GAG/GTTTGT), which when strengthened to a consensus sequence (GAG/GTAAGT) leads to near-total exon 16 inclusion. Impaired RBFOX2 binding reduces exon 16 inclusion in the context of the native weak 5′ splice site, but not the engineered strong 5′ splice site, implying that RBFOX2 achieves its effect by promoting utilization of the weak 5′ splice site. We further demonstrate that RBFOX2 increases U1 snRNP recruitment to the weak 5′ splice site through direct interaction between its C-terminal domain (CTD) and the zinc finger region of U1C and that the CTD is required for the effect of RBFOX2 on exon 16 splicing. Our data suggest a novel mechanism for exon 16 5′ splice site activation in which the binding of RBFOX2 to downstream intronic splicing enhancers stabilizes the pre-mRNA–U1 snRNP complex through interactions with U1C. PMID:22083953

  8. Xist Exon 7 Contributes to the Stable Localization of Xist RNA on the Inactive X-Chromosome

    PubMed Central

    Yamada, Norishige; Hasegawa, Yuko; Yue, Minghui; Hamada, Tomofumi; Nakagawa, Shinichi; Ogawa, Yuya

    2015-01-01

    To equalize X-linked gene dosage between the sexes in mammalian females, Xist RNA inactivates one of the two X-chromosomes. Here, we report the crucial function of Xist exon 7 in X-inactivation. Xist exon 7 is the second-largest exon with a well-conserved repeat E in eutherian mammals, but its role is often overlooked in X-inactivation. Although female ES cells with a targeted truncation of the Xist exon 7 showed no significant differences in their Xist expression levels and RNA stability from control cells expressing wild-type Xist, compromised localization of Xist RNA and incomplete silencing of X-linked genes on the inactive X-chromosome (Xi) were observed in the exon 7-truncated mutant cells. Furthermore, the interaction between the mutant Xist RNA and hnRNP U required for localization of Xist RNA to the Xi was impaired in the Xist exon 7 truncation mutant cells. Our results suggest that exon 7 of Xist RNA plays an important role for stable Xist RNA localization and silencing of the X-linked genes on the Xi, possibly acting through an interaction with hnRNP U. PMID:26244333

  9. Identification and qualification of 500 nuclear, single-copy, orthologous genes for the Eupulmonata (Gastropoda) using transcriptome sequencing and exon capture.

    PubMed

    Teasdale, Luisa C; Köhler, Frank; Murray, Kevin D; O'Hara, Tim; Moussalli, Adnan

    2016-09-01

    The qualification of orthology is a significant challenge when developing large, multiloci phylogenetic data sets from assembled transcripts. Transcriptome assemblies have various attributes, such as fragmentation, frameshifts and mis-indexing, which pose problems to automated methods of orthology assessment. Here, we identify a set of orthologous single-copy genes from transcriptome assemblies for the land snails and slugs (Eupulmonata) using a thorough approach to orthology determination involving manual alignment curation, gene tree assessment and sequencing from genomic DNA. We qualified the orthology of 500 nuclear, protein-coding genes from the transcriptome assemblies of 21 eupulmonate species to produce the most complete phylogenetic data matrix for a major molluscan lineage to date, both in terms of taxon and character completeness. Exon capture targeting 490 of the 500 genes (those with at least one exon >120 bp) from 22 species of Australian Camaenidae successfully captured sequences of 2825 exons (representing all targeted genes), with only a 3.7% reduction in the data matrix due to the presence of putative paralogs or pseudogenes. The automated pipeline Agalma retrieved the majority of the manually qualified 500 single-copy gene set and identified a further 375 putative single-copy genes, although it failed to account for fragmented transcripts resulting in lower data matrix completeness when considering the original 500 genes. This could potentially explain the minor inconsistencies we observed in the supported topologies for the 21 eupulmonate species between the manually curated and 'Agalma-equivalent' data set (sharing 458 genes). Overall, our study confirms the utility of the 500 gene set to resolve phylogenetic relationships at a range of evolutionary depths and highlights the importance of addressing fragmentation at the homolog alignment stage for probe design. PMID:27289081

  10. Hypokalemic paralysis due to thyrotoxicosis accompanied by Gitelman's syndrome.

    PubMed

    Baldane, S; Ipekci, S H; Celik, S; Gundogdu, A; Kebapcilar, L

    2015-01-01

    A 35-year-old male patient was admitted with fatigue and muscle weakness. He had been on methimazole due to thyrotoxicosis for 2 weeks. Laboratory tests showed overt hyperthyroidism and hypokalemia. Potassium replacement was started with an initial diagnosis of thyrotoxic hypokalemic periodic paralysis. Later on, despite the euthyroid condition and potassium chloride treatment, hypokalemia persisted. Further investigations revealed hyperreninemic hyperaldosteronism. The patient was considered to have Gitelman's syndrome (GS) and all genetic analysis was done. A c. 1145C>T, p. Thr382Met homozygote missense mutation located on solute carrier family 12, member gene 3, exon 9 was detected and GS was confirmed. PMID:25838649

  11. Hypokalemic paralysis due to thyrotoxicosis accompanied by Gitelman's syndrome

    PubMed Central

    Baldane, S.; Ipekci, S. H.; Celik, S.; Gundogdu, A.; Kebapcilar, L.

    2015-01-01

    A 35-year-old male patient was admitted with fatigue and muscle weakness. He had been on methimazole due to thyrotoxicosis for 2 weeks. Laboratory tests showed overt hyperthyroidism and hypokalemia. Potassium replacement was started with an initial diagnosis of thyrotoxic hypokalemic periodic paralysis. Later on, despite the euthyroid condition and potassium chloride treatment, hypokalemia persisted. Further investigations revealed hyperreninemic hyperaldosteronism. The patient was considered to have Gitelman's syndrome (GS) and all genetic analysis was done. A c. 1145C>T, p. Thr382Met homozygote missense mutation located on solute carrier family 12, member gene 3, exon 9 was detected and GS was confirmed. PMID:25838649

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

    PubMed Central

    Hernández-Imaz, Elisabete; Martín, Yolanda; de Conti, Laura; Melean, German; Valero, Ana; Baralle, Marco; Hernández-Chico, Concepción

    2015-01-01

    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. PMID:26509978

  13. Systematic discovery of regulated and conserved alternative exons in the mammalian brain reveals NMD modulating chromatin regulators.

    PubMed

    Yan, Qinghong; Weyn-Vanhentenryck, Sebastien M; Wu, Jie; Sloan, Steven A; Zhang, Ye; Chen, Kenian; Wu, Jia Qian; Barres, Ben A; Zhang, Chaolin

    2015-03-17

    Alternative splicing (AS) dramatically expands the complexity of the mammalian brain transcriptome, but its atlas remains incomplete. Here we performed deep mRNA sequencing of mouse cortex to discover and characterize alternative exons with potential functional significance. Our analysis expands the list of AS events over 10-fold compared with previous annotations, demonstrating that 72% of multiexon genes express multiple splice variants in this single tissue. To evaluate functionality of the newly discovered AS events, we conducted comprehensive analyses on central nervous system (CNS) cell type-specific splicing, targets of tissue- or cell type-specific RNA binding proteins (RBPs), evolutionary selection pressure, and coupling of AS with nonsense-mediated decay (AS-NMD). We show that newly discovered events account for 23-42% of all cassette exons under tissue- or cell type-specific regulation. Furthermore, over 7,000 cassette exons are under evolutionary selection for regulated AS in mammals, 70% of which are new. Among these are 3,058 highly conserved cassette exons, including 1,014 NMD exons that may function directly to control gene expression levels. These NMD exons are particularly enriched in RBPs including splicing factors and interestingly also regulators for other steps of RNA metabolism. Unexpectedly, a second group of NMD exons reside in genes encoding chromatin regulators. Although the conservation of NMD exons in RBPs frequently extends into lower vertebrates, NMD exons in chromatin regulators are introduced later into the mammalian lineage, implying the emergence of a novel mechanism coupling AS and epigenetics. Our results highlight previously uncharacterized complexity and evolution in the mammalian brain transcriptome. PMID:25737549

  14. Response to MET inhibitors in patients with stage IV lung adenocarcinomas harboring MET mutations causing exon 14 skipping

    PubMed Central

    Paik, Paul K.; Drilon, Alexander; Fan, Pang-Dian; Yu, Helena; Rekhtman, Natasha; Ginsberg, Michelle S.; Borsu, Laetitia; Schultz, Nikolaus; Berger, Michael F.; Rudin, Charles M.; Ladanyi, Marc

    2015-01-01

    Mutations in the MET exon 14 RNA splice acceptor and donor sites, which lead to exon skipping, deletion of the juxtamembrane domain containing the Cbl E3-ubiquitin ligase binding site, and decreased turnover of the resultant aberrant MET protein, were previously reported to be oncogenic in preclinical models. We now report responses to the MET inhibitors crizotinib and cabozantinib in four patients with stage IV lung adenocarcinomas harboring mutations leading to MET exon 14 skipping, highlighting a new therapeutic strategy for the 4% of lung adenocarcinoma patients whose tumors harbor this previously underappreciated genetic alteration. PMID:25971939

  15. Large exonic deletions in POLR3B gene cause POLR3-related leukodystrophy.

    PubMed

    Gutierrez, Mariana; Thiffault, Isabelle; Guerrero, Kether; Martos-Moreno, Gabriel Á; Tran, Luan T; Benko, William; van der Knaap, Marjo S; van Spaendonk, Rosalina M L; Wolf, Nicole I; Bernard, Geneviève

    2015-01-01

    POLR3-related (or 4H) leukodystrophy is an autosomal recessive disorder caused by mutations in POLR3A or POLR3B and is characterized by neurological and non-neurological features. In a small proportion of patients, no mutation in either gene or only one mutation is found. Analysis of the POLR3B cDNA revealed a large deletion of exons 21-22 in one case and of exons 26-27 in another case. These are the first reports of long deletions causing POLR3-related leukodystrophy, suggesting that deletions and duplications in POLR3A or POLR3B should be investigated in patients with a compatible phenotype, especially if one pathogenic variant has been identified. PMID:26045207

  16. Variable intron/exon structure in the oligochaete lombricine kinase gene.

    PubMed

    Doumen, Chris

    2012-09-01

    Lombricine kinase is an annelid enzyme that belongs to the phosphagen kinase family of which creatine kinase and arginine kinase are the typical representatives. The enzymes play important roles in the cellular energy metabolism of animals. Biochemical, physiological and molecular information with respect to lombricine kinase is limited compared to other phosphagen kinases. This study presents data on the cDNA sequences of lombricine kinase from two smaller oligochaetes, Enchytraeus sp. and Stylaria sp. The deduced amino acid sequences are analyzed and compared with other selected phosphagen kinases. The intron/exon structure of the lombricine kinase gene was determined for these two species as well as two additional oligochaetes, Lumbriculus variegatus and Tubifex tubifex, and compared with available data for annelid phosphagen kinases. The data indicate the existence of a variable organization of the proposed 8-intron/9-exon gene structure. The results provide further insights in the evolution and position of these enzymes within the phosphagen kinase family. PMID:22705027

  17. BRCA1 EXON 11, a CERES (composite regulatory element of splicing) element involved in splice regulation.

    PubMed

    Tammaro, Claudia; Raponi, Michela; Wilson, David I; Baralle, Diana

    2014-01-01

    Unclassified variants (UV) of BRCA1 can affect normal pre-mRNA splicing. Here, we investigate the UV c.693G>A, a "silent" change in BRCA1 exon 11, which we have found induces aberrant splicing in patient carriers and in vitro. Using a minigene assay, we show that the UV c.693G>A has a strong effect on the splicing isoform ratio of BRCA1. Systematic site-directed mutagenesis of the area surrounding the nucleotide position c.693G>A induced variable changes in the level of exon 11 inclusion/exclusion in the mRNA, pointing to the presence of a complex regulatory element with overlapping enhancer and silencer functions. Accordingly, protein binding analysis in the region detected several splicing regulatory factors involved, including SRSF1, SRSF6 and SRSF9, suggesting that this sequence represents a composite regulatory element of splicing (CERES). PMID:25056543

  18. The mouse formin (Fmn) gene: Genomic structure, novel exons, and genetic mapping

    SciTech Connect

    Wang, C.C.; Chan, D.C.; Leder, P.

    1997-02-01

    Mutations in the mouse formin (Fmn) gene, formerly known as the limb deformity (ld) gene, give rise to recessively inherited limb deformities and renal malformations or aplasia. The Fmn gene encodes many differentially processed transcripts that are expressed in both adult and embryonic tissues. To study the genomic organization of the Fmn locus, we have used Fmn probes to isolate and characterize genomic clones spanning 500 kb. Our analysis of these clones shows that the Fmn gene is composed of at least 24 exons and spans 400 kb. We have identified two novel exons that are expressed in the developing embryonic limb bud as well as adult tissues such as brain and kidney. We have also used a microsatellite polymorphism from within the Fmn gene to map it genetically to a 2.2-cM interval between D2Mit58 and D2Mit103. 36 refs., 6 figs., 1 tab.

  19. Exon-intron structure of the human neuronal nicotinic acetylcholine receptor {alpha}4 subunit (CHRNA4)

    SciTech Connect

    Steinlein, O.; Weiland, S.; Stoodt, J.; Propping, P.

    1996-03-01

    The human neuronal nicotinic acetylcholine receptor {alpha}4 subunit gene (CHRNA4) is located in the candidate region for three different phenotypes: benign familial neonatal convulsions, autosomal dominant nocturnal frontal lobe epilepsy, and low-voltage EEG. Recently, a missense mutation in transmembrane domain 2 of CHRNA4 was found to be associated with autosomal dominant nocturnal frontal lobe epilepsy in one extended pedigree. We have determined the genomic organization of CHRNA4, which consists of six exons distributed over approximately 17 kb of genomic DNA. The nucleotide sequence obtained from the genomic regions adjacent to the exon boundaries enabled us to develop a set of primer pairs for PCR amplification of the complete coding region. The sequence analysis provides the basis for a comprehensive mutation screening of CHRNA4 in the above-mentioned phenotypes and possibly in other types of idopathic epilepsies. 29 refs., 3 figs., 1 tab.

  20. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice.

    PubMed

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J A; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose-fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  1. Histone H3 lysine 9 trimethylation and HP1γ favor inclusion of alternative exons.

    PubMed

    Saint-André, Violaine; Batsché, Eric; Rachez, Christophe; Muchardt, Christian

    2011-03-01

    Pre-messenger RNAs (pre-mRNAs) maturation is initiated cotranscriptionally. It is therefore conceivable that chromatin-borne information participates in alternative splicing. Here we find that elevated levels of trimethylation of histone H3 on Lys9 (H3K9me3) are a characteristic of the alternative exons of several genes including CD44. On this gene the chromodomain protein HP1γ, frequently defined as a transcriptional repressor, facilitates inclusion of the alternative exons via a mechanism involving decreased RNA polymerase II elongation rate. In addition, accumulation of HP1γ on the variant region of the CD44 gene stabilizes association of the pre-mRNA with the chromatin. Altogether, our data provide evidence for localized histone modifications impacting alternative splicing. They further implicate HP1γ as a possible bridging molecule between the chromatin and the maturating mRNA, with a general impact on splicing decisions. PMID:21358630

  2. Recognition of alternatively spliced cassette exons based on a hybrid model.

    PubMed

    Zhang, Xiaokang; Peng, Qinke; Li, Liang; Li, Xintong

    2016-03-11

    Alternative splicing (AS) is an important mechanism of gene regulation that contributes to protein diversity. It is of great significance to recognize different kinds of AS accurately so as to understand the mechanism of gene regulation. Many in silico methods have been applied to detecting AS with vast features, but the result is far from satisfactory. In this paper, we used the features proven to be useful in recognizing AS in previous literature and proposed a hybrid method combining Gene Expression Programming (GEP) and Random Forests (RF) to classify the constitutive exons and cassette exons which is the most common AS phenomenon. GEP will firstly make prediction to the samples of strong signal, and the other samples of weak signal will be distinguished with a more complex classifier based on RF. The experiment result indicates that this method can highly improve the recognition level in this issue. PMID:26869516

  3. Hexose enhances oligonucleotide delivery and exon skipping in dystrophin-deficient mdx mice

    PubMed Central

    Han, Gang; Gu, Ben; Cao, Limin; Gao, Xianjun; Wang, Qingsong; Seow, Yiqi; Zhang, Ning; Wood, Matthew J. A.; Yin, HaiFang

    2016-01-01

    Carbohydrate-based infusion solutions are widely used in the clinic. Here we show that co-administration of phosphorodiamidate morpholino oligomers (PMOs) with glucose enhances exon-skipping activity in Duchenne muscular dystrophy (DMD) mdx mice. We identify a glucose–fructose (GF) formulation that potentiates PMO activity, completely corrects aberrant Dmd transcripts, restores dystrophin levels in skeletal muscles and achieves functional rescue without detectable toxicity. This activity is attributed to enhancement of GF-mediated PMO uptake in the muscle. We demonstrate that PMO cellular uptake is energy dependent, and that ATP from GF metabolism contributes to enhanced cellular uptake of PMO in the muscle. Collectively, we show that GF potentiates PMO activity by replenishing cellular energy stores under energy-deficient conditions in mdx mice. Our findings provide mechanistic insight into hexose-mediated oligonucleotide delivery and have important implications for the development of DMD exon-skipping therapy. PMID:26964641

  4. Selective Blockade of Periostin Exon 17 Preserves Cardiac Performance in Acute Myocardial Infarction.

    PubMed

    Taniyama, Yoshiaki; Katsuragi, Naruto; Sanada, Fumihiro; Azuma, Junya; Iekushi, Kazuma; Koibuchi, Nobutaka; Okayama, Keita; Ikeda-Iwabu, Yuka; Muratsu, Jun; Otsu, Rei; Rakugi, Hiromi; Morishita, Ryuichi

    2016-02-01

    We previously reported that overexpression of full-length periostin, Pn-1, resulted in ventricular dilation with enhanced interstitial collagen deposition in a rat model. However, other reports have documented that the short-form splice variants Pn-2 (lacking exon 17) and Pn-4 (lacking exons 17 and 21) promoted cardiac repair by angiogenesis and prevented cardiac rupture after acute myocardial infarction. The apparently differing findings from those reports prompted us to use a neutralizing antibody to selectively inhibit Pn-1 by blockade of exon 17 in a rat acute myocardial infarction model. Administration of Pn neutralizing antibody resulted in a significant decrease in the infarcted and fibrotic areas of the myocardium, which prevented ventricular wall thinning and dilatation. The inhibition of fibrosis by Pn neutralizing antibody was associated with a significant decrease in gene expression of fibrotic markers, including collagen I, collagen III, and transforming growth factor-β1. Importantly, the number of α-smooth muscle actin-positive myofibroblasts was significantly reduced in the hearts of animals treated with Pn neutralizing antibody, whereas cardiomyocyte proliferation and angiogenesis were comparable in the IgG and neutralizing antibody groups. Moreover, the level of Pn-1 expression was significantly correlated with the severity of myocardial infarction. In addition, Pn-1, but not Pn-2 or Pn-4, inhibited fibroblast and myocyte attachment, which might account for the cell slippage observed during cardiac remodeling. Collectively, these results indicate that therapeutics that specifically inhibit Pn exon-17, via a neutralizing antibody or drug, without suppressing other periostin variants might offer a new class of medication for the treatment of acute myocardial infarction patients. PMID:26644236

  5. Towards a transcription map of human chromosome 21: Identification of expressed sequences by exon trapping

    SciTech Connect

    Chen, H.M.; Chrast, R.; Rossier, C.

    1994-09-01

    Chromosome 21q contains about 1% of the human genome, and when triplicated is responsible for Down syndrome. The genetic and physical maps of this chromosome are amongst the most developed of all human chromosomes. A considerable international effort is now under way with the aims of cloning and mapping all chromosome 21 genes, assigning functions, and determining their involvement in disease phenotypes. We have used exon trapping/amplification methods to identify exons of genes that map on chromosome 21. EcoR1 or Bam HI-digested DNA from pools of 96 cosmids from the chromosome 21 library LL21NC02{open_quotes}Q{close_quotes} were used for cloning in vector pSLP3 (after elimination of cosmids positive for ribosomal RNR genes and mouse DNA); recombinant plasmids were transfected into cos7 cells and trapped sequences were subcloned. False positive clones, i.e. those containing vector self-spliced sequences (which represented between 8-30% of clones in different experiments), have been eliminated by hybridization of oligonucleotides corresponding to sequences of the vector self-spliced events. More than 100 different trapped {open_quotes}exons{close_quotes} have been identified to date after single or double pass sequencing. Two sequences matched exons of known genes on chromosome 21 (COL6A 1 and MX1). About 45% of the sequences were entirely new, i.e. there was no homology with entries in the nucleotide or protein databases (blastin and blastx searches). An additional 48% of the sequences were homologous but not identical to sequences in the databases. Only 4% were repetitive elements. Specific homologies will be presented. All of the trapped sequences that have been mapped by filter hybridization, PCR, or FISH, map back to cosmids or YACs of chromosome 21. This approach permits rapid identification of expressed sequences of this chromosome, the cloning of its genes, and the understanding of its disorders.

  6. A homozygous deletion of exon 1 in WISP3 causes progressive pseudorheumatoid dysplasia in two siblings

    PubMed Central

    Neerinckx, Barbara; Thues, Cedric; Wouters, Carine; Lechner, Sarah; Westhovens, Rene; Van Esch, Hilde

    2015-01-01

    Progressive pseudorheumatoid dysplasia (PPD) is a rare autosomal recessive disease that causes progressive joint stiffness and pain. It is associated with loss-of-function mutations in the WISP3 gene. We describe two sisters suffering from PPD in whom molecular genetic analysis revealed a homozygous deletion of exon 1 and of the 5′UTR of the WISP3 gene. This is the first time that a gross deletion has been described as the causal mutation in PPD. PMID:27081554

  7. Functional and Structural Consequence of Rare Exonic Single Nucleotide Polymorphisms: One Story, Two Tales

    PubMed Central

    Gu, Wanjun; Gurguis, Christopher I.; Zhou, Jin J.; Zhu, Yihua; Ko, Eun-A.; Ko, Jae-Hong; Wang, Ting; Zhou, Tong

    2015-01-01

    Genetic variation arising from single nucleotide polymorphisms (SNPs) is ubiquitously found among human populations. While disease-causing variants are known in some cases, identifying functional or causative variants for most human diseases remains a challenging task. Rare SNPs, rather than common ones, are thought to be more important in the pathology of most human diseases. We propose that rare SNPs should be divided into two categories dependent on whether the minor alleles are derived or ancestral. Derived alleles are less likely to have been purified by evolutionary processes and may be more likely to induce deleterious effects. We therefore hypothesized that the rare SNPs with derived minor alleles would be more important for human diseases and predicted that these variants would have larger functional or structural consequences relative to the rare variants for which the minor alleles are ancestral. We systematically investigated the consequences of the exonic SNPs on protein function, mRNA structure, and translation. We found that the functional and structural consequences are more significant for the rare exonic variants for which the minor alleles are derived. However, this pattern is reversed when the minor alleles are ancestral. Thus, the rare exonic SNPs with derived minor alleles are more likely to be deleterious. Age estimation of rare SNPs confirms that these potentially deleterious SNPs are recently evolved in the human population. These results have important implications for understanding the function of genetic variations in human exonic regions and for prioritizing functional SNPs in genome-wide association studies of human diseases. PMID:26454016

  8. A novel exon 3 mutation in a Tunisian patient with Lafora's disease.

    PubMed

    Khiari, H Mrabet; Lesca, G; Malafosse, A; Mrabet, A

    2011-05-15

    We report a Tunisian patient born from consanguineous marriage affected with progressive myoclonus epilepsy and cognitive decline, consistent with the diagnosis of Lafora disease. Genetic analysis showed a novel c.659 T>A mutation on exon 3 of the EPM2A gene, converting a leucine to a glutamine residue at amino acid position 220 (p.Leu220Gln), in the dual-specificity phosphatase domain. PMID:21371719

  9. DVL1 Frameshift Mutations Clustering in the Penultimate Exon Cause Autosomal-Dominant Robinow Syndrome

    PubMed Central

    White, Janson; Mazzeu, Juliana F.; Hoischen, Alexander; Jhangiani, Shalini N.; Gambin, Tomasz; Alcino, Michele Calijorne; Penney, Samantha; Saraiva, Jorge M.; Hove, Hanne; Skovby, Flemming; Kayserili, Hülya; Estrella, Elicia; Vulto-van Silfhout, Anneke T.; Steehouwer, Marloes; Muzny, Donna M.; Sutton, V. Reid; Gibbs, Richard A.; Lupski, James R.; Brunner, Han G.; van Bon, Bregje W.M.; Carvalho, Claudia M.B.

    2015-01-01

    Robinow syndrome is a genetically heterogeneous disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features and for which both autosomal-recessive and autosomal-dominant inheritance patterns have been described. Causative variants in the non-canonical signaling gene WNT5A underlie a subset of autosomal-dominant Robinow syndrome (DRS) cases, but most individuals with DRS remain without a molecular diagnosis. We performed whole-exome sequencing in four unrelated DRS-affected individuals without coding mutations in WNT5A and found heterozygous DVL1 exon 14 mutations in three of them. Targeted Sanger sequencing in additional subjects with DRS uncovered DVL1 exon 14 mutations in five individuals, including a pair of monozygotic twins. In total, six distinct frameshift mutations were found in eight subjects, and all were heterozygous truncating variants within the penultimate exon of DVL1. In five families in which samples from unaffected parents were available, the variants were demonstrated to represent de novo mutations. All variant alleles are predicted to result in a premature termination codon within the last exon, escape nonsense-mediated decay (NMD), and most likely generate a C-terminally truncated protein with a distinct −1 reading-frame terminus. Study of the transcripts extracted from affected subjects’ leukocytes confirmed expression of both wild-type and variant alleles, supporting the hypothesis that mutant mRNA escapes NMD. Genomic variants identified in our study suggest that truncation of the C-terminal domain of DVL1, a protein hypothesized to have a downstream role in the Wnt-5a non-canonical pathway, is a common cause of DRS. PMID:25817016

  10. DVL1 frameshift mutations clustering in the penultimate exon cause autosomal-dominant Robinow syndrome.

    PubMed

    White, Janson; Mazzeu, Juliana F; Hoischen, Alexander; Jhangiani, Shalini N; Gambin, Tomasz; Alcino, Michele Calijorne; Penney, Samantha; Saraiva, Jorge M; Hove, Hanne; Skovby, Flemming; Kayserili, Hülya; Estrella, Elicia; Vulto-van Silfhout, Anneke T; Steehouwer, Marloes; Muzny, Donna M; Sutton, V Reid; Gibbs, Richard A; Lupski, James R; Brunner, Han G; van Bon, Bregje W M; Carvalho, Claudia M B

    2015-04-01

    Robinow syndrome is a genetically heterogeneous disorder characterized by mesomelic limb shortening, genital hypoplasia, and distinctive facial features and for which both autosomal-recessive and autosomal-dominant inheritance patterns have been described. Causative variants in the non-canonical signaling gene WNT5A underlie a subset of autosomal-dominant Robinow syndrome (DRS) cases, but most individuals with DRS remain without a molecular diagnosis. We performed whole-exome sequencing in four unrelated DRS-affected individuals without coding mutations in WNT5A and found heterozygous DVL1 exon 14 mutations in three of them. Targeted Sanger sequencing in additional subjects with DRS uncovered DVL1 exon 14 mutations in five individuals, including a pair of monozygotic twins. In total, six distinct frameshift mutations were found in eight subjects, and all were heterozygous truncating variants within the penultimate exon of DVL1. In five families in which samples from unaffected parents were available, the variants were demonstrated to represent de novo mutations. All variant alleles are predicted to result in a premature termination codon within the last exon, escape nonsense-mediated decay (NMD), and most likely generate a C-terminally truncated protein with a distinct -1 reading-frame terminus. Study of the transcripts extracted from affected subjects' leukocytes confirmed expression of both wild-type and variant alleles, supporting the hypothesis that mutant mRNA escapes NMD. Genomic variants identified in our study suggest that truncation of the C-terminal domain of DVL1, a protein hypothesized to have a downstream role in the Wnt-5a non-canonical pathway, is a common cause of DRS. PMID:25817016

  11. First Report of a Single Exon Deletion in TCOF1 Causing Treacher Collins Syndrome

    PubMed Central

    Beygo, J.; Buiting, K.; Seland, S.; Lüdecke, H.-J.; Hehr, U.; Lich, C.; Prager, B.; Lohmann, D.R.; Wieczorek, D.

    2012-01-01

    Treacher Collins syndrome (TCS) is a rare craniofacial disorder characterized by facial anomalies and ear defects. TCS is caused by mutations in the TCOF1 gene and follows autosomal dominant inheritance. Recently, mutations in the POLR1D and POLR1C genes have also been identified to cause TCS. However, in a subset of patients no causative mutation could be found yet. Inter- and intrafamilial phenotypic variability is high as is the variety of mainly family-specific mutations identified throughout TCOF1. No obvious correlation between pheno- and genotype could be observed. The majority of described point mutations, small insertions and deletions comprising only a few nucleotides within TCOF1 lead to a premature termination codon. We investigated a cohort of 112 patients with a tentative clinical diagnosis of TCS by multiplex ligation-dependent probe amplification (MLPA) to search for larger deletions not detectable with other methods used. All patients were selected after negative screening for mutations in TCOF1, POLR1D and POLR1C. In 1 patient with an unequivocal clinical diagnosis of TCS, we identified a 3.367 kb deletion. This deletion abolishes exon 3 and is the first described single exon deletion within TCOF1. On RNA level we observed loss of this exon which supposedly leads to haploinsufficiency of TREACLE, the nucleolar phosphoprotein encoded by TCOF1. PMID:22712005

  12. Plug-and-Play Genetic Access to Drosophila Cell Types Using Exchangeable Exon Cassettes

    PubMed Central

    Diao, Fengqiu; Ironfield, Holly; Luan, Haojiang; Diao, Feici; Shropshire, William C.; Ewer, John; Marr, Elizabeth; Potter, Christopher J.; Landgraf, Matthias; White, Benjamin H.

    2015-01-01

    Summary Genetically encoded effectors are important tools for probing cellular function in living animals, but improved methods for directing their expression to specific cell types are required. Here we introduce a simple, versatile method for achieving cell type-specific expression of transgenes that leverages the untapped potential of “coding introns” (i.e. introns between coding exons). Our method couples the expression of a transgene to that of a native gene expressed in the cells of interest using intronically inserted “plug-and-play” cassettes (called “Trojan exons”) that carry a splice acceptor site followed by the coding sequences of T2A peptide and an effector transgene. We demonstrate the efficacy of this approach in Drosophila using lines containing suitable MiMIC transposons and a palette of Trojan exons capable of expressing a range of commonly used transcription factors. We also introduce an exchangeable, MiMIC-like Trojan exon construct that can be targeted to coding introns using the Crispr/Cas system. PMID:25732830

  13. Subfunctionalization of duplicate mitf genes associated with differential degeneration of alternative exons in fish.

    PubMed Central

    Altschmied, Joachim; Delfgaauw, Jacqueline; Wilde, Brigitta; Duschl, Jutta; Bouneau, Laurence; Volff, Jean-Nicolas; Schartl, Manfred

    2002-01-01

    The microphthalmia-associated transcription factor (MITF) exists in at least four isoforms. These are generated in higher vertebrates using alternative 5' exons and promoters from a single gene. Two separate genes (mitf-m and mitf-b), however, are present in different teleost fish species including the poeciliid Xiphophorus, the pufferfishes Fugu rubripes and Tetraodon nigroviridis, and the zebrafish Danio rerio. Fish proteins MITF-m and MITF-b correspond at both the structural and the expression levels to one particular bird/mammalian MITF isoform. In the teleost lineage subfunctionalization of mitf genes after duplication at least 100 million years ago is associated with the degeneration of alternative exons and, probably, regulatory elements and promoters. For example, a remnant of the first exon specific for MITF-m is detected within the pufferfish gene encoding MITF-b. Retracing the evolutionary history of mitf genes in vertebrates uncovered the differential recruitment of new introns specific for either the teleost or the bird/mammalian lineage. PMID:12019239

  14. Representation of DNA sequences in genetic codon context with applications in exon and intron prediction.

    PubMed

    Yin, Changchuan

    2015-04-01

    To apply digital signal processing (DSP) methods to analyze DNA sequences, the sequences first must be specially mapped into numerical sequences. Thus, effective numerical mappings of DNA sequences play key roles in the effectiveness of DSP-based methods such as exon prediction. Despite numerous mappings of symbolic DNA sequences to numerical series, the existing mapping methods do not include the genetic coding features of DNA sequences. We present a novel numerical representation of DNA sequences using genetic codon context (GCC) in which the numerical values are optimized by simulation annealing to maximize the 3-periodicity signal to noise ratio (SNR). The optimized GCC representation is then applied in exon and intron prediction by Short-Time Fourier Transform (STFT) approach. The results show the GCC method enhances the SNR values of exon sequences and thus increases the accuracy of predicting protein coding regions in genomes compared with the commonly used 4D binary representation. In addition, this study offers a novel way to reveal specific features of DNA sequences by optimizing numerical mappings of symbolic DNA sequences. PMID:25491390

  15. mRNA-Associated Processes and Their Influence on Exon-Intron Structure in Drosophila melanogaster

    PubMed Central

    Lepennetier, Gildas; Catania, Francesco

    2016-01-01

    mRNA-associated processes and gene structure in eukaryotes are typically treated as separate research subjects. Here, we bridge this separation and leverage the extensive multidisciplinary work on Drosophila melanogaster to examine the roles that capping, splicing, cleavage/polyadenylation, and telescripting (i.e., the protection of nascent transcripts from premature cleavage/polyadenylation by the splicing factor U1) might play in shaping exon-intron architecture in protein-coding genes. Our findings suggest that the distance between subsequent internal 5′ splice sites (5′ss) in Drosophila genes is constrained such that telescripting effects are maximized, in theory, and thus nascent transcripts are less vulnerable to premature termination. Exceptionally weak 5′ss and constraints on intron-exon size at the gene 5′ end also indicate that capping might enhance the recruitment of U1 and, in turn, promote telescripting at this location. Finally, a positive correlation between last exon length and last 5′ss strength suggests that optimal donor splice sites in the proximity of the pre-mRNA tail may inhibit the processing of downstream polyadenylation signals more than weak donor splice sites do. These findings corroborate and build upon previous experimental and computational studies on Drosophila genes. They support the possibility, hitherto scantly explored, that mRNA-associated processes impose significant constraints on the evolution of eukaryotic gene structure. PMID:27172210

  16. NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis

    PubMed Central

    Dolata, Jakub; Guo, Yanwu; Kołowerzo, Agnieszka; Smoliński, Dariusz; Brzyżek, Grzegorz; Jarmołowski, Artur; Świeżewski, Szymon

    2015-01-01

    The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5′ and 3′ splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co-localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1-dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis. PMID:25568310

  17. Combined analysis of exon splicing and genome wide polymorphism data predict schizophrenia risk loci.

    PubMed

    Oldmeadow, Christopher; Mossman, David; Evans, Tiffany-Jane; Holliday, Elizabeth G; Tooney, Paul A; Cairns, Murray J; Wu, Jingqin; Carr, Vaughan; Attia, John R; Scott, Rodney J

    2014-05-01

    Schizophrenia has a strong genetic basis, and genome-wide association studies (GWAS) have shown that effect sizes for individual genetic variants which increase disease risk are small, making detection and validation of true disease-associated risk variants extremely challenging. Specifically, we first identify genes with exons showing differential expression between cases and controls, indicating a splicing mechanism that may contribute to variation in disease risk and focus on those showing consistent differential expression between blood and brain tissue. We then perform a genome-wide screen for SNPs associated with both normalised exon intensity of these genes (so called splicing QTLs) as well as association with schizophrenia. We identified a number of significantly associated loci with a biologically plausible role in schizophrenia, including MCPH1, DLG3, ZC3H13, and BICD2, and additional loci that influence splicing of these genes, including YWHAH. Our approach of integrating genome-wide exon intensity with genome-wide polymorphism data has identified a number of plausible SZ associated loci. PMID:24507884

  18. Two-Exon Skipping within MLPH Is Associated with Coat Color Dilution in Rabbits

    PubMed Central

    Lehner, Stefanie; Gähle, Marion; Dierks, Claudia; Stelter, Ricarda; Gerber, Jonathan; Brehm, Ralph; Distl, Ottmar

    2013-01-01

    Coat color dilution turns black coat color to blue and red color to cream and is a characteristic in many mammalian species. Matings among Netherland Dwarf, Loh, and Lionhead Dwarf rabbits over two generations gave evidence for a monogenic autosomal recessive inheritance of coat colour dilution. Histological analyses showed non-uniformly distributed, large, agglomerating melanin granules in the hair bulbs of coat color diluted rabbits. We sequenced the cDNA of MLPH in two dilute and one black rabbit for polymorphism detection. In both color diluted rabbits, skipping of exons 3 and 4 was present resulting in altered amino acids at p.QGL[37-39]QWA and a premature stop codon at p.K40*. Sequencing of genomic DNA revealed a c.111-5C>A splice acceptor mutation within the polypyrimidine tract of intron 2 within MLPH. This mutation presumably causes skipping of exons 3 and 4. In 14/15 dilute rabbits, the c.111-5C>A mutation was homozygous and in a further dilute rabbit, heterozygous and in combination with a homozygous frame shift mutation within exon 6 (c.585delG). In conclusion, our results demonstrated a colour dilution associated MLPH splice variant causing a strongly truncated protein (p.Q37QfsX4). An involvement of further MLPH-associated mutations needs further investigations. PMID:24376820

  19. Absence of regulated splicing of fibronectin EDA exon reduces atherosclerosis in mice

    PubMed Central

    Babaev, Vladimir R.; Porro, Fabiola; Linton, MacRae F.; Fazio, Sergio; Baralle, Francisco E.; Muro, Andrés F.

    2008-01-01

    Atherosclerotic lesions are characterized by a profound alteration in the architecture of the arterial intima, with a marked increase of fibronectin (FN) and the appearance of the alternatively spliced FN variant containing the Extra Domain A (EDA). To analyze the role of FN isoforms in atherosclerotic lesion formation we utilized mouse strains devoid of EDA-exon regulated splicing, which constitutively include (EDA+/+) or exclude (EDA−/−) the exon. Both mutant mice had a 40% reduction in atherosclerotic lesions after the atherogenic-diet treatment (Mean±SE, μm2; 22969±2185; 13660±1533; 14260±2501 for EDAwt/wt, EDA+/+ and EDA−/−, respectively; p≤0.01 ANOVA test) associated to a lower capacity of macrophages to uptake modified LDL and undergo foam-cell formation. Lesions in control mice were more numerous and bigger, with augmented and deeper macrophage infiltration, and increased FN expression in the sub-endothelial area. Previous experiments have shown that apoE−/−EDA−/− mice have a decreased number and size of atherosclerotic lesions and, on this basis, it has been proposed that the EDA domain has a pro-atherogenic role. Our data with the EDA+/+ mice rules out this hypothesis and suggest that regulated splicing of the EDA exon of the FN gene is involved in progression of atherosclerosis, highlighting the importance of alternative splicing in regulating cellular processes. PMID:17897651

  20. Intracellular Folding of the Tetrahymena Group I Intron Depends on Exon Sequence and Promoter Choice

    SciTech Connect

    Koduvayur,S.; Woodson, S.

    2004-01-01

    The Tetrahymena group I intron splices 20 to 50 times faster in Tetrahymena than in vitro, implying that the intron rapidly adopts its active conformation in the cell. The importance of cotranscriptional folding and the contribution of the rRNA exons to the stability of the active pre-RNA structure were investigated by comparing the activity of minimal pre-RNAs expressed in Escherichia coli. Pre-RNAs containing exons derived from E. coli 23 S rRNA were three to four times more active than the wild-type Tetrahymena pre-RNA. E. coli transcripts of the chimeric E. coli pre-RNA were two to eight times more active than were T7 transcripts. However, the effect of cotranscriptional folding depends on exon sequences. Unexpectedly, the unspliced pre-RNA decays more slowly than predicted from the rate of splicing. This observation is best explained by partitioning of transcripts into active and inactive pools. We propose that the active pool splices within a few seconds, whereas the inactive pool is degraded without appreciable splicing.

  1. Calpain cleavage within dysferlin exon 40a releases a synaptotagmin-like module for membrane repair

    PubMed Central

    Redpath, G. M. I.; Woolger, N.; Piper, A. K.; Lemckert, F. A.; Lek, A.; Greer, P. A.; North, K. N.; Cooper, S. T.

    2014-01-01

    Dysferlin and calpain are important mediators of the emergency response to repair plasma membrane injury. Our previous research revealed that membrane injury induces cleavage of dysferlin to release a synaptotagmin-like C-terminal module we termed mini-dysferlinC72. Here we show that injury-activated cleavage of dysferlin is mediated by the ubiquitous calpains via a cleavage motif encoded by alternately spliced exon 40a. An exon 40a–specific antibody recognizing cleaved mini-dysferlinC72 intensely labels the circumference of injury sites, supporting a key role for dysferlinExon40a isoforms in membrane repair and consistent with our evidence suggesting that the calpain-cleaved C-terminal module is the form specifically recruited to injury sites. Calpain cleavage of dysferlin is a ubiquitous response to membrane injury in multiple cell lineages and occurs independently of the membrane repair protein MG53. Our study links calpain and dysferlin in the calcium-activated vesicle fusion of membrane repair, placing calpains as upstream mediators of a membrane repair cascade that elicits cleaved dysferlin as an effector. Of importance, we reveal that myoferlin and otoferlin are also cleaved enzymatically to release similar C-terminal modules, bearing two C2 domains and a transmembrane domain. Evolutionary preservation of this feature highlights its functional importance and suggests that this highly conserved C-terminal region of ferlins represents a functionally specialized vesicle fusion module. PMID:25143396

  2. Antisense-mediated Exon Skipping Decreases Tau Protein Expression: A Potential Therapy For Tauopathies

    PubMed Central

    Sud, Reeteka; Geller, Evan T; Schellenberg, Gerard D

    2014-01-01

    In Alzheimer's disease, progressive supranuclear palsy, and a number of other neurodegenerative diseases, the microtubule associated protein tau aggregates to form intracellular neurofibrillary tangles and glial tangles, abnormal structures that are part of disease pathogenesis. Disorders with aggregated tau are called tauopathies. Presently, there are no disease-modifying treatments for this disease class. Tau is encoded by the MAPT gene. We propose that reducing MAPT expression and thus the amount of tau protein made could prevent aggregation, and potentially be an approach to treat tauopathies. We tested 31 morpholinos, complementary to the sense strand of the MAPT gene to identify oligonucleotides that can downregulate MAPT expression and reduce the amount of tau protein produced. Oligonucleotides were tested in human neuroblastoma cell lines SH-SY5Y and IMR32. We identified several morpholinos that reduced MAPT mRNA expression up to 50% and tau protein levels up to ~80%. The two most potent oligonucleotides spanned the 3′ boundary of exons 1 and 5, masking the 5′-splice sites of these exons. Both morpholinos induced skipping of the targeted exons. These in vitro findings were confirmed in mice transgenic for the entire human MAPT gene and that express human tau protein. These studies demonstrate the feasibility of using modified oligonucleotides to alter tau expression. PMID:25072694

  3. mRNA-Associated Processes and Their Influence on Exon-Intron Structure in Drosophila melanogaster.

    PubMed

    Lepennetier, Gildas; Catania, Francesco

    2016-01-01

    mRNA-associated processes and gene structure in eukaryotes are typically treated as separate research subjects. Here, we bridge this separation and leverage the extensive multidisciplinary work on Drosophila melanogaster to examine the roles that capping, splicing, cleavage/polyadenylation, and telescripting (i.e, the protection of nascent transcripts from premature cleavage/polyadenylation by the splicing factor U1) might play in shaping exon-intron architecture in protein-coding genes. Our findings suggest that the distance between subsequent internal 5' splice sites (5'ss) in Drosophila genes is constrained such that telescripting effects are maximized, in theory, and thus nascent transcripts are less vulnerable to premature termination. Exceptionally weak 5'ss and constraints on intron-exon size at the gene 5' end also indicate that capping might enhance the recruitment of U1 and, in turn, promote telescripting at this location. Finally, a positive correlation between last exon length and last 5'ss strength suggests that optimal donor splice sites in the proximity of the pre-mRNA tail may inhibit the processing of downstream polyadenylation signals more than weak donor splice sites do. These findings corroborate and build upon previous experimental and computational studies on Drosophila genes. They support the possibility, hitherto scantly explored, that mRNA-associated processes impose significant constraints on the evolution of eukaryotic gene structure. PMID:27172210

  4. Han Chinese patients with dopa-responsive dystonia exhibit a low frequency of exonic deletion in the GCH1 gene.

    PubMed

    Shi, W T; Cai, C Y; Li, M S; Ling, C; Li, W D

    2015-01-01

    We identified three novel mutations of the GTP cyclohydrolase 1 (GCH1) gene in patients with familial dopa-responsive dystonia (DRD), but were unable to identify meaningful sporadic mutations in patients with no obvious family DRD background. To investigate whether GCH1 regional deletions account for the etiology of DRD, we screened for heterozygous exonic deletions in DRD families and in patients with sporadic DRD. Multiple ligation-dependent probe amplification analysis and quantitative real-time polymerase chain reaction amplification was performed in all members of our DRD cohort and in controls to detect exonic deletions in GCH1, tyrosine hydroxylase, and the epsilon-sarcoglycan-encoding (SGCE) genes. Using these techniques, we detected a GCH1 exon 1 heterozygous deletion in 1 of 10 patients with sporadic DRD. Therefore, we concluded that exonic deletion in the GCH1 gene only accounted for the etiology in a small percentage of patients with sporadic DRD in our Han Chinese cohort. PMID:26400349

  5. The expression level of small non-coding RNAs derived from the first exon of protein-coding genes is predictive of cancer status

    PubMed Central

    Zovoilis, Athanasios; Mungall, Andrew J; Moore, Richard; Varhol, Richard; Chu, Andy; Wong, Tina; Marra, Marco; Jones, Steven JM

    2014-01-01

    Small non-coding RNAs (smRNAs) are known to be significantly enriched near the transcriptional start sites of genes. However, the functional relevance of these smRNAs remains unclear, and they have not been associated with human disease. Within the cancer genome atlas project (TCGA), we have generated small RNA datasets for many tumor types. In prior cancer studies, these RNAs have been regarded as transcriptional “noise,” due to their apparent chaotic distribution. In contrast, we demonstrate their striking potential to distinguish efficiently between cancer and normal tissues and classify patients with cancer to subgroups of distinct survival outcomes. This potential to predict cancer status is restricted to a subset of these smRNAs, which is encoded within the first exon of genes, highly enriched within CpG islands and negatively correlated with DNA methylation levels. Thus, our data show that genome-wide changes in the expression levels of small non-coding RNAs within first exons are associated with cancer. PMID:24534129

  6. Increased THEMIS First Exon Usage in CD4+ T-Cells Is Associated with a Genotype that Is Protective against Multiple Sclerosis

    PubMed Central

    Thompson, Sara; Kaur-Sandhu, Harpreet; Sawcer, Stephen; Coles, Alasdair; Ban, Maria; Jones, Joanne

    2016-01-01

    Multiple sclerosis is an autoimmune disease of the central nervous system. Genome wide association studies have identified over 100 common variants associated with multiple sclerosis, the majority of which implicate immunologically relevant genes, particularly those involved in T-cell development. SNP rs13204742 at the THEMIS/PTPRK locus is one such variant. Here, we have demonstrated mutually exclusive use of exon 1 and 2 amongst 16 novel THEMIS isoforms. We also show inverse correlation between THEMIS expression in human CD4+ T-cells and dosage of the multiple sclerosis risk allele at rs13204742, driven by reduced expression of exon 1- containing isoforms. In silico analysis suggests that this may be due to cell-specific, allele-dependent binding of the transcription factors FoxP3 and/or E47. Research exploring the functional implications of GWAS variants is important for gaining an understanding of disease pathogenesis, with the ultimate aim of identifying new therapeutic targets. PMID:27438997

  7. An evaluation of transcriptome-based exon capture for frog phylogenomics across multiple scales of divergence (Class: Amphibia, Order: Anura).

    PubMed

    Portik, Daniel M; Smith, Lydia L; Bi, Ke

    2016-09-01

    Custom sequence capture experiments are becoming an efficient approach for gathering large sets of orthologous markers in nonmodel organisms. Transcriptome-based exon capture utilizes transcript sequences to design capture probes, typically using a reference genome to identify intron-exon boundaries to exclude shorter exons (<200 bp). Here, we test directly using transcript sequences for probe design, which are often composed of multiple exons of varying lengths. Using 1260 orthologous transcripts, we conducted sequence captures across multiple phylogenetic scales for frogs, including outgroups ~100 Myr divergent from the ingroup. We recovered a large phylogenomic data set consisting of sequence alignments for 1047 of the 1260 transcriptome-based loci (~561 000 bp) and a large quantity of highly variable regions flanking the exons in transcripts (~70 000 bp), the latter improving substantially by only including ingroup species (~797 000 bp). We recovered both shorter (<100 bp) and longer exons (>200 bp), with no major reduction in coverage towards the ends of exons. We observed significant differences in the performance of blocking oligos for target enrichment and nontarget depletion during captures, and differences in PCR duplication rates resulting from the number of individuals pooled for capture reactions. We explicitly tested the effects of phylogenetic distance on capture sensitivity, specificity, and missing data, and provide a baseline estimate of expectations for these metrics based on a priori knowledge of nuclear pairwise differences among samples. We provide recommendations for transcriptome-based exon capture design based on our results, cost estimates and offer multiple pipelines for data assembly and analysis. PMID:27241806

  8. In silico screening based on predictive algorithms as a design tool for exon skipping oligonucleotides in Duchenne muscular dystrophy.

    PubMed

    Echigoya, Yusuke; Mouly, Vincent; Garcia, Luis; Yokota, Toshifumi; Duddy, William

    2015-01-01

    The use of antisense 'splice-switching' oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2'O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R² 0.89) and 53 (R² 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each position of

  9. Triple-layer dissection of the lung adenocarcinoma transcriptome: regulation at the gene, transcript, and exon levels.

    PubMed

    Hsu, Min-Kung; Wu, I-Ching; Cheng, Ching-Chia; Su, Jen-Liang; Hsieh, Chang-Huain; Lin, Yeong-Shin; Chen, Feng-Chi

    2015-10-01

    Lung adenocarcinoma is one of the most deadly human diseases. However, the molecular mechanisms underlying this disease, particularly RNA splicing, have remained underexplored. Here, we report a triple-level (gene-, transcript-, and exon-level) analysis of lung adenocarcinoma transcriptomes from 77 paired tumor and normal tissues, as well as an analysis pipeline to overcome genetic variability for accurate differentiation between tumor and normal tissues. We report three major results. First, more than 5,000 differentially expressed transcripts/exonic regions occur repeatedly in lung adenocarcinoma patients. These transcripts/exonic regions are enriched in nicotine metabolism and ribosomal functions in addition to the pathways enriched for differentially expressed genes (cell cycle, extracellular matrix receptor interaction, and axon guidance). Second, classification models based on rationally selected transcripts or exonic regions can reach accuracies of 0.93 to 1.00 in differentiating tumor from normal tissues. Of the 28 selected exonic regions, 26 regions correspond to alternative exons located in such regulators as tumor suppressor (GDF10), signal receptor (LYVE1), vascular-specific regulator (RASIP1), ubiquitination mediator (RNF5), and transcriptional repressor (TRIM27). Third, classification systems based on 13 to 14 differentially expressed genes yield accuracies near 100%. Genes selected by both detection methods include C16orf59, DAP3, ETV4, GABARAPL1, PPAR, RADIL, RSPO1, SERTM1, SRPK1, ST6GALNAC6, and TNXB. Our findings imply a multilayered lung adenocarcinoma regulome in which transcript-/exon-level regulation may be dissociated from gene-level regulation. Our described method may be used to identify potentially important genes/transcripts/exonic regions for the tumorigenesis of lung adenocarcinoma and to construct accurate tumor vs. normal classification systems for this disease. PMID:26356813

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

    PubMed Central

    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

  11. In Silico Screening Based on Predictive Algorithms as a Design Tool for Exon Skipping Oligonucleotides in Duchenne Muscular Dystrophy

    PubMed Central

    Echigoya, Yusuke; Mouly, Vincent; Garcia, Luis; Yokota, Toshifumi; Duddy, William

    2015-01-01

    The use of antisense ‘splice-switching’ oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD), for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1) the binding energetics of the oligonucleotide to the RNA, and (2) the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many) into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted) and/or 2’O Methyl RNA oligonucleotides (76% correctly predicted). Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R2 0.89) and 53 (R2 0.89), one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each

  12. Triple-layer dissection of the lung adenocarcinoma transcriptome – regulation at the gene, transcript, and exon levels

    PubMed Central

    Hsu, Min-Kung; Wu, I-Ching; Cheng, Ching-Chia; Su, Jen-Liang; Hsieh, Chang-Huain; Lin, Yeong-Shin; Chen, Feng-Chi

    2015-01-01

    Lung adenocarcinoma is one of the most deadly human diseases. However, the molecular mechanisms underlying this disease, particularly RNA splicing, have remained underexplored. Here, we report a triple-level (gene-, transcript-, and exon-level) analysis of lung adenocarcinoma transcriptomes from 77 paired tumor and normal tissues, as well as an analysis pipeline to overcome genetic variability for accurate differentiation between tumor and normal tissues. We report three major results. First, more than 5,000 differentially expressed transcripts/exonic regions occur repeatedly in lung adenocarcinoma patients. These transcripts/exonic regions are enriched in nicotine metabolism and ribosomal functions in addition to the pathways enriched for differentially expressed genes (cell cycle, extracellular matrix receptor interaction, and axon guidance). Second, classification models based on rationally selected transcripts or exonic regions can reach accuracies of 0.93 to 1.00 in differentiating tumor from normal tissues. Of the 28 selected exonic regions, 26 regions correspond to alternative exons located in such regulators as tumor suppressor (GDF10), signal receptor (LYVE1), vascular-specific regulator (RASIP1), ubiquitination mediator (RNF5), and transcriptional repressor (TRIM27). Third, classification systems based on 13 to 14 differentially expressed genes yield accuracies near 100%. Genes selected by both detection methods include C16orf59, DAP3, ETV4, GABARAPL1, PPAR, RADIL, RSPO1, SERTM1, SRPK1, ST6GALNAC6, and TNXB. Our findings imply a multilayered lung adenocarcinoma regulome in which transcript-/exon-level regulation may be dissociated from gene-level regulation. Our described method may be used to identify potentially important genes/transcripts/exonic regions for the tumorigenesis of lung adenocarcinoma and to construct accurate tumor vs. normal classification systems for this disease. PMID:26356813

  13. An exon 53 frameshift mutation in CUBN abrogates cubam function and causes Imerslund-Gräsbeck syndrome in dogs.

    PubMed

    Fyfe, John C; Hemker, Shelby L; Venta, Patrick J; Fitzgerald, Caitlin A; Outerbridge, Catherine A; Myers, Sherry L; Giger, Urs

    2013-08-01

    Cobalamin malabsorption accompanied by selective proteinuria is an autosomal recessive disorder known as Imerslund-Gräsbeck syndrome in humans and was previously described in dogs due to amnionless (AMN) mutations. The resultant vitamin B12 deficiency causes dyshematopoiesis, lethargy, failure to thrive, and life-threatening metabolic disruption in the juvenile period. We studied 3 kindreds of border collies with cobalamin malabsorption and mapped the disease locus in affected dogs to a 2.9Mb region of homozygosity on canine chromosome 2. The region included CUBN, the locus encoding cubilin, a peripheral membrane protein that in concert with AMN forms the functional intrinsic factor-cobalamin receptor expressed in ileum and a multi-ligand receptor in renal proximal tubules. Cobalamin malabsorption and proteinuria comprising CUBN ligands were demonstrated by radiolabeled cobalamin uptake studies and SDS-PAGE, respectively. CUBN mRNA and protein expression were reduced ~10 fold and ~20 fold, respectively, in both ileum and kidney of affected dogs. DNA sequencing demonstrated a single base deletion in exon 53 predicting a translational frameshift and early termination codon likely triggering nonsense mediated mRNA decay. The mutant allele segregated with the disease in the border collie kindred. The border collie disorder indicates that a CUBN mutation far C-terminal from the intrinsic factor-cobalamin binding site can abrogate receptor expression and cause Imerslund-Gräsbeck syndrome. PMID:23746554

  14. A silent exonic SNP in kdm3a affects nucleic acids structure but does not regulate experimental autoimmune encephalomyelitis.

    PubMed

    Gillett, Alan; Bergman, Petra; Parsa, Roham; Bremges, Andreas; Giegerich, Robert; Jagodic, Maja

    2013-01-01

    Defining genetic variants that predispose for diseases is an important initiative that can improve biological understanding and focus therapeutic development. Genetic mapping in humans and animal models has defined genomic regions controlling a variety of phenotypes known as quantitative trait loci (QTL). Causative disease determinants, including single nucleotide polymorphisms (SNPs), lie within these regions and can often be identified through effects on gene expression. We previously identified a QTL on rat chromosome 4 regulating macrophage phenotypes and immune-mediated diseases including experimental autoimmune encephalomyelitis (EAE). Gene analysis and a literature search identified lysine-specific demethylase 3A (Kdm3a) as a potential regulator of these phenotypes. Genomic sequencing determined only two synonymous SNPs in Kdm3a. The silent synonymous SNP in exon 15 of Kdm3a caused problems with quantitative PCR detection in the susceptible strain through reduced amplification efficiency due to altered secondary cDNA structure. Shape Probability Shift analysis predicted that the SNP often affects RNA folding; thus, it may impact protein translation. Despite these differences in rats, genetic knockout of Kdm3a in mice resulted in no dramatic effect on immune system development and activation or EAE susceptibility and severity. These results provide support for tools that analyze causative SNPs that impact nucleic acid structures. PMID:24312603

  15. Cooperative binding of TIA-1 and U1 snRNP in K-SAM exon splicing activation

    SciTech Connect

    Gesnel, Marie-Claude; Theoleyre, Sandrine; Del Gatto-Konczak, Fabienne; Breathnach, Richard . E-mail: breathna@nantes.inserm.fr

    2007-07-13

    In 293 cells, splicing of the human fibroblast growth factor receptor-2 K-SAM alternative exon is inefficient, but can be made efficient by provoking TIA-1 binding to the U-rich IAS1 sequence downstream from the exon's 5' splice site. We show here that TIA-1 domains known to interact with U1 snRNP and to recruit it to 5' splice sites in vitro are required for TIA-1 activation of K-SAM exon splicing in vivo. We further show that tethering downstream from the K-SAM exon a fusion between the U1 snRNP component U1C and the bacteriophage MS2 coat protein provokes IAS1-dependent exon splicing, and present evidence that the fusion functions after its incorporation into U1 snRNP. Our in vivo data, taken together with previous in vitro results, show that K-SAM splicing activation involves cooperative binding of TIA-1 and U1 snRNP to the exon's 5' splice site region.

  16. The splicing regulatory element, UGCAUG, is phylogenetically and spatially conserved in introns that flank tissue-specific alternative exons

    PubMed Central

    Minovitsky, Simon; Gee, Sherry L.; Schokrpur, Shiruyeh; Dubchak, Inna; Conboy, John G.

    2005-01-01

    Previous studies have identified UGCAUG as an intron splicing enhancer that is frequently located adjacent to tissue-specific alternative exons in the human genome. Here, we show that UGCAUG is phylogenetically and spatially conserved in introns that flank brain-enriched alternative exons from fish to man. Analysis of sequence from the mouse, rat, dog, chicken and pufferfish genomes revealed a strongly statistically significant association of UGCAUG with the proximal intron region downstream of brain-enriched alternative exons. The number, position and sequence context of intronic UGCAUG elements were highly conserved among mammals and in chicken, but more divergent in fish. Control datasets, including constitutive exons and non-tissue-specific alternative exons, exhibited a much lower incidence of closely linked UGCAUG elements. We propose that the high sequence specificity of the UGCAUG element, and its unique association with tissue-specific alternative exons, mark it as a critical component of splicing switch mechanism(s) designed to activate a limited repertoire of splicing events in cell type-specific patterns. We further speculate that highly conserved UGCAUG-binding protein(s) related to the recently described Fox-1 splicing factor play a critical role in mediating this specificity. PMID:15691898

  17. Extensive and prolonged restoration of dystrophin expression with vivo-morpholino-mediated multiple exon skipping in dystrophic dogs.

    PubMed

    Yokota, Toshifumi; Nakamura, Akinori; Nagata, Tetsuya; Saito, Takashi; Kobayashi, Masanori; Aoki, Yoshitsugu; Echigoya, Yusuke; Partridge, Terence; Hoffman, Eric P; Takeda, Shin'ichi

    2012-10-01

    Duchenne muscular dystrophy (DMD) is a severe and the most prevalent form of muscular dystrophy, characterized by rapid progression of muscle degeneration. Antisense-mediated exon skipping is currently one of the most promising therapeutic options for DMD. However, unmodified antisense oligos such as morpholinos require frequent (weekly or bi-weekly) injections. Recently, new generation morpholinos such as vivo-morpholinos are reported to lead to extensive and prolonged dystrophin expression in the dystrophic mdx mouse, an animal model of DMD. The vivo-morpholino contains a cell-penetrating moiety, octa-guanidine dendrimer. Here, we sought to test the efficacy of multiple exon skipping of exons 6-8 with vivo-morpholinos in the canine X-linked muscular dystrophy, which harbors a splice site mutation at the boundary of intron 6 and exon 7. We designed and optimized novel antisense cocktail sequences and combinations for exon 8 skipping and demonstrated effective exon skipping in dystrophic dogs in vivo. Intramuscular injections with newly designed cocktail oligos led to high levels of dystrophin expression, with some samples similar to wild-type levels. This is the first report of successful rescue of dystrophin expression with morpholino conjugates in dystrophic dogs. Our results show the potential of phosphorodiamidate morpholino oligomer conjugates as therapeutic agents for DMD. PMID:22888777

  18. Effective quantitative real-time polymerase chain reaction analysis of the parkin gene (PARK2) exon 1–12 dosage

    PubMed Central

    Shadrina, Maria I; Semenova, Elena V; Slominsky, Petr A; Bagyeva, Gulbahar H; Illarioshkin, Sergei N; Ivanova-Smolenskaia, Irina I; Limborska, Svetlana A

    2007-01-01

    Background One of the causes of Parkinson's disease is mutations in the PARK2 gene. Deletions and duplications of single exons or exon groups account for a large proportion of the gene mutations. Direct detection of these mutations can be used for the diagnosis of Parkinson's disease. Methods To detect these mutations, we developed an effective technique based on the real-time TaqMan PCR system, which allows us to evaluate the copynumbers of the PARK2 gene exons by comparing the intensity of the amplification signals from some exon of this gene with that of the β-globin gene (the internal control). Results We analyzed rearrangements in exons 1–12 of the PARK2 gene in 64 patients from Russia with early-onset Parkinson's disease. The frequency of these mutations in our patients was 14%. Conclusion We have developed a simple, accurate, and reproducible method applicable to the rapid detection of exon rearrangements in the PARK2 gene. It is suitable for the analysis of large patient groups, and it may become the basis for a diagnostic test. PMID:17324265

  19. Wild-Type Mouse Models to Screen Antisense Oligonucleotides for Exon-Skipping Efficacy in Duchenne Muscular Dystrophy

    PubMed Central

    Cao, Limin; Han, Gang; Gu, Ben; Yin, HaiFang

    2014-01-01

    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. PMID:25365558

  20. Differences among lesions with exon 19, exon 21 EGFR mutations and wild types in surgically resected non-small cell lung cancer

    PubMed Central

    Jin, Ying; Chen, Ming; Yu, Xinmin

    2016-01-01

    The clinical behavior of patients with advanced non-small cell lung cancer (NSCLC) differ between epidermal growth factor receptor (EGFR) exon 19 deletion (Ex19) and EGFR exon 21 L858R mutation (Ex21). This study aimed to evaluate whether these differences exist in surgically resected NSCLC. A total of 198 patients with surgically resected NSCLC harbouring Ex19 (n = 53), Ex21 (n = 51), and EGFR wild-type (Wt) (n = 94) were analyzed. The clinicopathological features, laboratory parameters, recurrent sites and disease-free survival (DFS) were compared according to mutational EGFR status. Ex21 occurred more frequently in female (p < 0.001), never-smokers (p < 0.001), adenocarcinoma (p < 0.001), low grade (p = 0.013) than Wt lesions. Ex19 occurred more frequently in female (p = 0.016), never-smokers (p = 0.008), adenocarcinoma (p < 0.001), low grade (p = 0.025) than Wt lesions. Ex 21 lesions (p = 0.026) had larger lepidic components than Wt lesions. Wt lesions had larger mucinous variant components than Ex21 lesions (p = 0.045) and Ex19 lesions (p = 0.015). Ex21 lesions were associated with lower pretreatment neutrophil: lymphocyte ratio (NLR) than Wt lesions (p = 0.017). The recurrent sites and DFS were similar among patients with Wt, Ex19 and Ex21. PMID:27527915

  1. Physiologically generated presenilin 1 lacking exon 8 fails to rescue brain PS1−/− phenotype and forms complexes with wildtype PS1 and nicastrin

    PubMed Central

    Brautigam, Hannah; Moreno, Cesar L.; Steele, John W.; Bogush, Alexey; Dickstein, Dara L.; Kwok, John B.J.; Schofield, Peter R.; Thinakaran, Gopal; Mathews, Paul M.; Hof, Patrick R.; Gandy, Sam; Ehrlich, Michelle E.

    2015-01-01

    The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer’s disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1∆exon8). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1∆exon8 reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1∆exon8 did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1∆exon8 is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1∆exon8 in vivo by crossing PS1∆exon8 transgenics with either PS1-null or Dutch APPE693Q mice. As a control, we crossed APPE693Q with mice expressing a deletion in an adjacent exon (PS1∆exon9). PS1∆exon8 did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1∆exon8 interacts with nicastrin, participating in the γ–secretase complex formation. These data support that catalytically inactive PS1∆exon8 is generated physiologically and participates in protein-protein interactions. PMID:26608390

  2. Physiologically generated presenilin 1 lacking exon 8 fails to rescue brain PS1-/- phenotype and forms complexes with wildtype PS1 and nicastrin.

    PubMed

    Brautigam, Hannah; Moreno, Cesar L; Steele, John W; Bogush, Alexey; Dickstein, Dara L; Kwok, John B J; Schofield, Peter R; Thinakaran, Gopal; Mathews, Paul M; Hof, Patrick R; Gandy, Sam; Ehrlich, Michelle E

    2015-01-01

    The presenilin 1 (PSEN1) L271V mutation causes early-onset familial Alzheimer's disease by disrupting the alternative splicing of the PSEN1 gene, producing some transcripts harboring the L271V point mutation and other transcripts lacking exon 8 (PS1(∆exon8)). We previously reported that PS1 L271V increased amyloid beta (Aβ) 42/40 ratios, while PS1(∆exon8) reduced Aβ42/40 ratios, indicating that the former and not the exon 8 deletion transcript is amyloidogenic. Also, PS1(∆exon8) did not rescue Aβ generation in PS1/2 double knockout cells indicating its identity as a severe loss-of-function splice form. PS1(∆exon8) is generated physiologically raising the possibility that we had identified the first physiological inactive PS1 isoform. We studied PS1(∆exon8) in vivo by crossing PS1(∆exon8) transgenics with either PS1-null or Dutch APP(E693Q) mice. As a control, we crossed APP(E693Q) with mice expressing a deletion in an adjacent exon (PS1(∆exon9)). PS1(∆exon8) did not rescue embryonic lethality or Notch-deficient phenotypes of PS1-null mice displaying severe loss of function in vivo. We also demonstrate that this splice form can interact with wildtype PS1 using cultured cells and co-immunoprecipitation (co-IP)/bimolecular fluorescence complementation. Further co-IP demonstrates that PS1(∆exon8) interacts with nicastrin, participating in the γ-secretase complex formation. These data support that catalytically inactive PS1(∆exon8) is generated physiologically and participates in protein-protein interactions. PMID:26608390

  3. Splicing defects in the COL3A1 gene: marked preference for 5' (donor) spice-site mutations in patients with exon-skipping mutations and Ehlers-Danlos syndrome type IV.

    PubMed Central

    Schwarze, U; Goldstein, J A; Byers, P H

    1997-01-01

    Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis. Images Figure 1 Figure 3 Figure 6 PMID:9399899

  4. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811+1.6kbA-->G, produces a new exon: high frequency in Spanish cystic fibrosis chromosomes and association with severe phenotype.

    PubMed Central

    Chillón, M; Dörk, T; Casals, T; Giménez, J; Fonknechten, N; Will, K; Ramos, D; Nunes, V; Estivill, X

    1995-01-01

    mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6kbA-->G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA-->G-mRNA was 5-10-fold less abundant than delta F508 mRNA. Mutation 1811+1.6kbA-->G was found in 21 Spanish and 1 German CF chromosomes, making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype delta F508/1811+1.6kbA-->G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. Images Figure 3 PMID:7534040

  5. A novel donor splice site in intron 11 of the CFTR gene, created by mutation 1811 + 1.6kbA {yields} G, produces a new exon: High frequency in spanish cystic fibrosis chromosomes and association with severe phenotype

    SciTech Connect

    Chillon, M.; Casals, T.; Gimenez, J.; Ramos, D.; Nunes, V.; Estivill, X.; Doerk, T.; Will, K.; Fonknechten, N.

    1995-03-01

    mRNA analysis of the cystic fibrosis transmembrane regulator (CFTR) gene in tissues of cystic fibrosis (CF) patients has allowed us to detect a cryptic exon. The new exon involves 49 base pairs between exons 11 and 12 and is due to a point mutation (1811+1.6bA{yields}G) that creates a new donor splice site in intron 11. Semiquantitative mRNA analysis showed that 1811+1.6kbA{r_arrow}G-mRNA was 5-10-fold less abundant than {triangle}F508 mRNA. Mutations 1811+1.6kbA{yields}G was found in 21 Spanish and 1 German CF chromosome(s), making it the fourth-most-frequent mutation (2%) in the Spanish population. Individuals with genotype {triangle}F508/1811+1.6kbA{yields}G have only 1%-3% of normal CFTR mRNA. This loss of 97% of normal CFTR mRNA must be responsible for the pancreatic insufficiency and for the severe CF phenotype in these patients. 30 refs., 3 figs., 2 tabs.

  6. The heparan sulphate deficient Hspg2 exon 3 null mouse displays reduced deposition of TGF-β1 in skin compared to C57BL/6 wild type mice.

    PubMed

    Shu, Cindy; Smith, Susan M; Melrose, James

    2016-06-01

    This was an observational study where we examined the role of perlecan HS on the deposition of TGF-β1 in C57BL/6 and Hspg2(∆3-/∆3-) perlecan exon 3 null mouse skin. Despite its obvious importance in skin repair and tissue homeostasis no definitive studies have immunolocalised TGF-β1 in skin in WT or Hspg2(∆3-/∆3-) perlecan exon 3 null mice. Vertical parasagittal murine dorsal skin from 3, 6 and 12 week old C57BL/6 and Hspg2(∆3-/∆3-) mice were fixed in neutral buffered formalin, paraffin embedded and 4 μm sections stained with Mayers haematoxylin and eosin (H & E). TGF-β1 was immunolocalised using a rabbit polyclonal antibody, heat retrieval and the Envision NovaRED detection system. Immunolocalisation of TGF-β1 differed markedly in C57BL/6 and Hspg2(∆3-/∆3-) mouse skin, ablation of exon 3 of Hspg2 resulted in a very severe reduction in the deposition of TGF-β1 in skin 3-12 weeks postnatally. The reduced deposition of TGF-β1 observed in the present study would be expected to impact detrimentally on the remodelling and healing capacity of skin in mutant mice compounding on the poor wound-healing properties already reported for perlecan exon 3 null mice due to an inability to signal with FGF-2 and promote angiogenic repair processes. TGF-β1 also has cell mediated effects in tissue homeostasis and matrix stabilisation a reduction in TGF-β1 deposition would therefore be expected to detrimentally impact on skin homeostasis in the perlecan mutant mice. PMID:27098652

  7. Deleting exon 55 from the nebulin gene induces severe muscle weakness in a mouse model for nemaline myopathy

    PubMed Central

    Ottenheijm, Coen A. C.; Buck, Danielle; de Winter, Josine M.; Ferrara, Claudia; Piroddi, Nicoletta; Tesi, Chiara; Jasper, Jeffrey R.; Malik, Fady I.; Meng, Hui; Stienen, Ger J. M.; Beggs, Alan H.; Labeit, Siegfried; Poggesi, Corrado; Lawlor, Michael W.

    2013-01-01

    Nebulin—a giant sarcomeric protein—plays a pivotal role in skeletal muscle contractility by specifying thin filament length and function. Although mutations in the gene encoding nebulin (NEB) are a frequent cause of nemaline myopathy, the most common non-dystrophic congenital myopathy, the mechanisms by which mutations in NEB cause muscle weakness remain largely unknown. To better understand these mechanisms, we have generated a mouse model in which Neb exon 55 is deleted (NebΔExon55) to replicate a founder mutation seen frequently in patients with nemaline myopathy with Ashkenazi Jewish heritage. NebΔExon55 mice are born close to Mendelian ratios, but show growth retardation after birth. Electron microscopy studies show nemaline bodies—a hallmark feature of nemaline myopathy—in muscle fibres from NebΔExon55 mice. Western blotting studies with nebulin-specific antibodies reveal reduced nebulin levels in muscle from NebΔExon55 mice, and immunofluorescence confocal microscopy studies with tropomodulin antibodies and phalloidin reveal that thin filament length is significantly reduced. In line with reduced thin filament length, the maximal force generating capacity of permeabilized muscle fibres and single myofibrils is reduced in NebΔExon55 mice with a more pronounced reduction at longer sarcomere lengths. Finally, in NebΔExon55 mice the regulation of contraction is impaired, as evidenced by marked changes in crossbridge cycling kinetics and by a reduction of the calcium sensitivity of force generation. A novel drug that facilitates calcium binding to the thin filament significantly augmented the calcium sensitivity of submaximal force to levels that exceed those observed in untreated control muscle. In conclusion, we have characterized the first nebulin-based nemaline myopathy model, which recapitulates important features of the phenotype observed in patients harbouring this particular mutation, and which has severe muscle weakness caused by thin

  8. A novel exon in the human Ca2+-activated Cl- channel Ano1 imparts greater sensitivity to intracellular Ca2.

    PubMed

    Strege, Peter R; Bernard, Cheryl E; Mazzone, Amelia; Linden, David R; Beyder, Arthur; Gibbons, Simon J; Farrugia, Gianrico

    2015-11-01

    Anoctamin 1 (Ano1; TMEM16A) is a Ca(2+)-activated Cl(-) channel (CACC) expressed in interstitial cells of Cajal. The mechanisms by which Ca(2+) regulates Ano1 are incompletely understood. In the gastrointestinal tract, Ano1 is required for normal slow wave activity and is involved in regulating cell proliferation. Splice variants of Ano1 have varying electrophysiological properties and altered expression in disease states. Recently, we identified a transcript for human Ano1 containing a novel exon-"exon 0" upstream of and in frame with exon 1. The electrophysiological properties of this longer Ano1 isoform are unknown. Our aim was to determine the functional contribution of the newly identified exon to the Ca(2+) sensitivity and electrophysiological properties of Ano1. Constructs with [Ano1(+0)] or without [Ano1(-0)] the newly identified exon were transfected into human embryonic kidney-293 cells. Voltage-clamp electrophysiology was used to determine voltage- and time-dependent parameters of whole cell Cl(-) currents between isoforms with varying concentrations of intracellular Ca(2+), extracellular anions, or Cl(-) channel inhibitors. We found that exon 0 did not change voltage sensitivity and had no impact on the relative permeability of Ano1 to most anions. Ano1(+0) exhibited greater changes in current density but lesser changes in kinetics than Ano1(-0) in response to varying intracellular Ca(2+). The CACC inhibitor niflumic acid inhibited current with greater efficacy and higher potency against Ano1(+0) compared with Ano1(-0). Likewise, the Ano1 inhibitor T16Ainh-A01 reduced Ano1(+0) more than Ano1(-0). In conclusion, human Ano1 containing exon 0 imparts its Cl(-) current with greater sensitivity to intracellular Ca(2+) and CACC inhibitors. PMID:26359375

  9. Follicle-stimulating hormone receptor (FSHR) alternative skipping of exon 2 or 3 affects ovarian response to FSH

    PubMed Central

    Karakaya, Cengiz; Guzeloglu-Kayisli, Ozlem; Hobbs, Rebecca J.; Gerasimova, Tsilya; Uyar, Asli; Erdem, Mehmet; Oktem, Mesut; Erdem, Ahmet; Gumuslu, Seyhan; Ercan, Deniz; Sakkas, Denny; Comizzoli, Pierre; Seli, Emre; Lalioti, Maria D.

    2014-01-01

    Genes critical for fertility are highly conserved in mammals. Interspecies DNA sequence variation, resulting in amino acid substitutions and post-transcriptional modifications, including alternative splicing, are a result of evolution and speciation. The mammalian follicle-stimulating hormone receptor (FSHR) gene encodes distinct species-specific forms by alternative splicing. Skipping of exon 2 of the human FSHR was reported in women of North American origin and correlated with low response to ovarian stimulation with exogenous follicle-stimulating hormone (FSH). To determine whether this variant correlated with low response in women of different genetic backgrounds, we performed a blinded retrospective observational study in a Turkish cohort. Ovarian response was determined as low, intermediate or high according to retrieved oocyte numbers after classifying patients in four age groups (<35, 35–37, 38–40, >40). Cumulus cells collected from 96 women undergoing IVF/ICSI following controlled ovarian hyperstimulation revealed four alternatively spliced FSHR products in seven patients (8%): exon 2 deletion in four patients; exon 3 and exons 2 + 3 deletion in one patient each, and a retention of an intron 1 fragment in one patient. In all others (92%) splicing was intact. Alternative skipping of exons 2, 3 or 2 + 3 were exclusive to low responders and was independent of the use of agonist or antagonist. Interestingly, skipping of exon 3 occurs naturally in the ovaries of domestic cats—a good comparative model for human fertility. We tested the signaling potential of human and cat variants after transfection in HEK293 cells and FSH stimulation. None of the splicing variants initiated cAMP signaling despite high FSH doses, unlike full-length proteins. These data substantiate the occurrence of FSHR exon skipping in a subgroup of low responders and suggest that species-specific regulation of FSHR splicing plays diverse roles in mammalian ovarian function. PMID

  10. MIR retroposon exonization promotes evolutionary variability and generates species-specific expression of IGF-1 splice variants.

    PubMed

    Annibalini, Giosuè; Bielli, Pamela; De Santi, Mauro; Agostini, Deborah; Guescini, Michele; Sisti, Davide; Contarelli, Serena; Brandi, Giorgio; Villarini, Anna; Stocchi, Vilberto; Sette, Claudio; Barbieri, Elena

    2016-05-01

    Insulin-like growth factor (IGF) -1 is a pleiotropic hormone exerting mitogenic and anti-apoptotic effects. Inclusion or exclusion of exon 5 into the IGF-1 mRNA gives rise to three transcripts, IGF-1Ea, IGF-1Eb and IGF-1Ec, which yield three different C-terminal extensions called Ea, Eb and Ec peptides. The biological significance of the IGF-1 splice variants and how the E-peptides affect the actions of mature IGF-1 are largely unknown. In this study we investigated the origin and conservation of the IGF-1 E-peptides and we compared the pattern of expression of the IGF-1 isoforms in vivo, in nine mammalian species, and in vitro using human and mouse IGF-1 minigenes. Our analysis showed that only IGF-1Ea is conserved among all vertebrates, whereas IGF-1Eb and IGF-1Ec are an evolutionary novelty originated from the exonization of a mammalian interspersed repetitive-b (MIR-b) element. Both IGF-1Eb and IGF-1Ec mRNAs were constitutively expressed in all mammalian species analyzed but their expression ratio varies greatly among species. Using IGF-1 minigenes we demonstrated that divergence in cis-acting regulatory elements between human and mouse conferred species-specific features to the exon 5 region. Finally, the protein-coding sequences of exon 5 showed low rate of synonymous mutations and contain disorder-promoting amino acids, suggesting a regulatory role for these domains. In conclusion, exonization of a MIR-b element in the IGF-1 gene determined gain of exon 5 during mammalian evolution. Alternative splicing of this novel exon added new regulatory elements at the mRNA and protein level potentially able to regulate the mature IGF-1 across tissues and species. PMID:27048986

  11. A point mutation within CD45 exon A is the cause of variant CD45RA splicing in humans.

    PubMed

    Zilch, C F; Walker, A M; Timón, M; Goff, L K; Wallace, D L; Beverley, P C

    1998-01-01

    The leukocyte common antigen (CD45) is alternatively spliced, generating various isoforms expressed on hemopoietic cells. The splicing pattern of CD45 in T cells is altered in some individuals who show abnormal expression of high molecular weight isoforms containing exon A. The variant splicing pattern was shown to be associated with heterozygosity for a silent point mutation within CD45 exon A. This C to G transition is located 77 nucleotides downstream of the splice acceptor junction of exon A (198 bp total length). Here we report that this mutation is the cause of abnormal splicing. To isolate the mutant gene, somatic cell hybrids of lymphocytes with a CD45 splicing defect and a mouse lymphoid line were produced and clones expressing different isoforms of CD45 were isolated. Expression of the high molecular weight isoform containing exon A was associated with the mutation within exon A. All hybrids expressing the low molecular weight isoforms lacking exon A contained the normal allele of CD45 only. In addition, minigenes including this mutation were constructed and transfected into various cell lines (COS-7, HeLa, CHO). Semi-quantitative reverse transcription polymerase chain reaction showed an increase of more than tenfold in splicing to CD45RA (concomitant with a decrease in splicing to CD45RO) when compared with the normal minigene. Taken together, these results demonstrate a causal relationship between the mutation in CD45 exon A and the variant splicing pattern observed. The involvement of trans-acting splicing factors that interact with this region of CD45 pre-mRNA is currently under investigation. PMID:9485182

  12. Vitamin D Receptor TaqI Gene Variant in Exon 9 and Polycystic Ovary Syndrome Risk

    PubMed Central

    Bagheri, Morteza; Abdi Rad, Isa; Hosseini Jazani, Nima; Nanbakhsh, Fariba

    2013-01-01

    Background: Polycystic ovary syndrome (PCOS) is known as a metabolic disorder. The results of recent studies implied that vitamin D receptor (VDR) genetic variants may impact PCOS and insulin resistance in women with PCOS. The aim of the present study was to determine the VDR TaqI gene variant in exon 9 (T/C) (rs731236) in normal controls and patients with PCOS for the first time in Iranian Azeri women. Materials and Methods: In this case control study between April 2011 and June 2012, a total of 76 women aged 18-40 years (38 patients with PCOS and 38 healthy women as normal controls) participated. Genotypes of VDR TaqI in exon 9 (T/C) (rs731236) were determined using the PCR-RFLP method. Results: The frequencies of VDR TaqI T anc C alleles were 0.605 and 0.395 in cases and 0.697 and 0.303 in controls. Also, the genotypic frequencies of VDR TaqI were 16) (42.11), 14(36.84), and 8(21.05) in cases, and 17(44.74), 19(50), and 2(5.26) in controls for TT, TC and CC genotypes respectively. There was no difference in genotype and allele frequencies between PCOS and controls (p value>0.05) with the exception of the CC genotype (p value=0.04). Conclusion: This report, a first of its own kind in Iranian Azeri patients, suggests that the CC genotype of VDR TaqI in exon 9 (rs731236) is associated with PCOS. PMID:24520473

  13. Sporadic ALS has compartment-specific aberrant exon splicing and altered cell–matrix adhesion biology

    PubMed Central

    Rabin, Stuart J.; Kim, Jae Mun ‘Hugo’; Baughn, Michael; Libby, Ryan T.; Kim, Young Joo; Fan, Yuxin; Libby, Randell T.; La Spada, Albert; Stone, Brad; Ravits, John

    2010-01-01

    Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by progressive weakness from loss of motor neurons. The fundamental pathogenic mechanisms are unknown and recent evidence is implicating a significant role for abnormal exon splicing and RNA processing. Using new comprehensive genomic technologies, we studied exon splicing directly in 12 sporadic ALS and 10 control lumbar spinal cords acquired by a rapid autopsy system that processed nervous systems specifically for genomic studies. ALS patients had rostral onset and caudally advancing disease and abundant residual motor neurons in this region. We created two RNA pools, one from motor neurons collected by laser capture microdissection and one from the surrounding anterior horns. From each, we isolated RNA, amplified mRNA, profiled whole-genome exon splicing, and applied advanced bioinformatics. We employed rigorous quality control measures at all steps and validated findings by qPCR. In the motor neuron enriched mRNA pool, we found two distinct cohorts of mRNA signals, most of which were up-regulated: 148 differentially expressed genes (P ≤ 10−3) and 411 aberrantly spliced genes (P ≤ 10−5). The aberrantly spliced genes were highly enriched in cell adhesion (P ≤ 10−57), especially cell–matrix as opposed to cell–cell adhesion. Most of the enriching genes encode transmembrane or secreted as opposed to nuclear or cytoplasmic proteins. The differentially expressed genes were not biologically enriched. In the anterior horn enriched mRNA pool, we could not clearly identify mRNA signals or biological enrichment. These findings, perturbed and up-regulated cell–matrix adhesion, suggest possible mechanisms for the contiguously progressive nature of motor neuron degeneration. Data deposition: GeneChip raw data (CEL-files) have been deposited for public access in the Gene Expression Omnibus (GEO), www.ncbi.nlm.nih.gov/geo, accession number GSE18920. PMID:19864493

  14. Exon Skipping in the RET Gene Encodes Novel Isoforms That Differentially Regulate RET Protein Signal Transduction.

    PubMed

    Gabreski, Nicole A; Vaghasia, Janki K; Novakova, Silvia S; McDonald, Neil Q; Pierchala, Brian A

    2016-07-29

    Rearranged during transfection (RET), a receptor tyrosine kinase that is activated by the glial cell line-derived neurotrophic factor family ligands (GFLs), plays a crucial role in the development and function of the nervous system and additionally is required for kidney development and spermatogenesis. RET encodes a transmembrane receptor that is 20 exons long and produces two known protein isoforms differing in C-terminal amino acid composition, referred to as RET9 and RET51. Studies of human pheochromocytomas identified two additional novel transcripts involving the skipping of exon 3 or exons 3, 4, and 5 and are referred to as RET(Δ) (E3) and RET(Δ) (E345), respectively. Here we report the presence of Ret(Δ) (E3) and Ret(Δ) (E345) in zebrafish, mice, and rats and show that these transcripts are dynamically expressed throughout development of the CNS, peripheral nervous system, and kidneys. We further explore the biochemical properties of these isoforms, demonstrating that, like full-length RET, RET(ΔE3) and RET(ΔE345) are trafficked to the cell surface, interact with all four GFRα co-receptors, and have the ability to heterodimerize with full-length RET. Signaling experiments indicate that RET(ΔE3) is phosphorylated in a similar manner to full-length RET. RET(ΔE345), in contrast, displays higher baseline autophosphorylation, specifically on the catalytic tyrosine, Tyr(905), and also on one of the most important signaling residues, Tyr(1062) These data provide the first evidence for a physiologic role of these isoforms in RET pathway function. PMID:27226544

  15. Mutation Scanning in Wheat by Exon Capture and Next-Generation Sequencing

    PubMed Central

    King, Robert; Bird, Nicholas; Ramirez-Gonzalez, Ricardo; Coghill, Jane A.; Patil, Archana; Hassani-Pak, Keywan; Uauy, Cristobal; Phillips, Andrew L.

    2015-01-01

    Targeted Induced Local Lesions in Genomes (TILLING) is a reverse genetics approach to identify novel sequence variation in genomes, with the aims of investigating gene function and/or developing useful alleles for breeding. Despite recent advances in wheat genomics, most current TILLING methods are low to medium in throughput, being based on PCR amplification of the target genes. We performed a pilot-scale evaluation of TILLING in wheat by next-generation sequencing through exon capture. An oligonucleotide-based enrichment array covering ~2 Mbp of wheat coding sequence was used to carry out exon capture and sequencing on three mutagenised lines of wheat containing previously-identified mutations in the TaGA20ox1 homoeologous genes. After testing different mapping algorithms and settings, candidate SNPs were identified by mapping to the IWGSC wheat Chromosome Survey Sequences. Where sequence data for all three homoeologues were found in the reference, mutant calls were unambiguous; however, where the reference lacked one or two of the homoeologues, captured reads from these genes were mis-mapped to other homoeologues, resulting either in dilution of the variant allele frequency or assignment of mutations to the wrong homoeologue. Competitive PCR assays were used to validate the putative SNPs and estimate cut-off levels for SNP filtering. At least 464 high-confidence SNPs were detected across the three mutagenized lines, including the three known alleles in TaGA20ox1, indicating a mutation rate of ~35 SNPs per Mb, similar to that estimated by PCR-based TILLING. This demonstrates the feasibility of using exon capture for genome re-sequencing as a method of mutation detection in polyploid wheat, but accurate mutation calling will require an improved genomic reference with more comprehensive coverage of homoeologues. PMID:26335335

  16. Targeted analysis of nucleotide and copy number variation by exon capture in allotetraploid wheat genome

    PubMed Central

    2011-01-01

    Background The ability of grass species to adapt to various habitats is attributed to the dynamic nature of their genomes, which have been shaped by multiple rounds of ancient and recent polyploidization. To gain a better understanding of the nature and extent of variation in functionally relevant regions of a polyploid genome, we developed a sequence capture assay to compare exonic sequences of allotetraploid wheat accessions. Results A sequence capture assay was designed for the targeted re-sequencing of 3.5 Mb exon regions that surveyed a total of 3,497 genes from allotetraploid wheat. These data were used to describe SNPs, copy number variation and homoeologous sequence divergence in coding regions. A procedure for variant discovery in the polyploid genome was developed and experimentally validated. About 1% and 24% of discovered SNPs were loss-of-function and non-synonymous mutations, respectively. Under-representation of replacement mutations was identified in several groups of genes involved in translation and metabolism. Gene duplications were predominant in a cultivated wheat accession, while more gene deletions than duplications were identified in wild wheat. Conclusions We demonstrate that, even though the level of sequence similarity between targeted polyploid genomes and capture baits can bias enrichment efficiency, exon capture is a powerful approach for variant discovery in polyploids. Our results suggest that allopolyploid wheat can accumulate new variation in coding regions at a high rate. This process has the potential to broaden functional diversity and generate new phenotypic variation that eventually can play a critical role in the origin of new adaptations and important agronomic traits. PMID:21917144

  17. A MAPT mutation in a regulatory element upstream of exon 10 causes frontotemporal dementia.

    PubMed

    Malkani, Roneil; D'Souza, Ian; Gwinn-Hardy, Katrina; Schellenberg, Gerard D; Hardy, John; Momeni, Parastoo

    2006-05-01

    We report here the genetic analysis of a newly ascertained kindred in which frontotemporal dementia occurs in an apparent autosomal dominant fashion, and in which a novel MAPT gene mutation co-segregates with disease. Sequencing the MAPT gene in affected individuals revealed a change in intron 9. This finding supports earlier studies on the effect of a splice-accepting element in inclusion of exon 10 in the MAPT transcript. This mutation sheds light on a novel mechanism by which over-expression of 4-repeat tau leads to disease. Based on our current findings, we propose a novel mechanism by which intronic mutations can lead to frontotemporal dementia. PMID:16503405

  18. ABERRANT SPLICING OF A BRAIN-ENRICHED ALTERNATIVE EXON ELIMINATES TUMOR SUPPRESSOR FUNCTION AND PROMOTES ONCOGENE FUNCTION DURING BRAIN TUMORIGENESIS

    PubMed Central

    Bredel, Markus; Ferrarese, Roberto; Harsh, Griffith R.; Yadav, Ajay K.; Bug, Eva; Maticzka, Daniel; Reichardt, Wilfried; Masilamani, Anie P.; Dai, Fangping; Kim, Hyunsoo; Hadler, Michael; Scholtens, Denise M.; Yu, Irene L.Y.; Beck, Jürgen; Srinivasasainagendra, Vinodh; Costa, Fabrizio; Baxan, Nicoleta; Pfeifer, Dietmar; Elverfeldt, Dominik v.; Backofen, Rolf; Weyerbrock, Astrid; Duarte, Christine W.; He, Xiaolin; Prinz, Marco; Chandler, James P.; Vogel, Hannes; Chakravarti, Arnab; Rich, Jeremy N.; Carro, Maria S.

    2014-01-01

    BACKGROUND: Tissue-specific alternative splicing is known to be critical to emergence of tissue identity during development, yet its role in malignant transformation is undefined. Tissue-specific splicing involves evolutionary-conserved, alternative exons, which represent only a minority of total alternative exons. Many, however, have functional features that influence activity in signaling pathways to profound biological effect. Given that tissue-specific splicing has a determinative role in brain development and the enrichment of genes containing tissue-specific exons for proteins with roles in signaling and development, it is thus plausible that changes in such exons could rewire normal neurogenesis towards malignant transformation. METHODS: We used integrated molecular genetic and cell biology analyses, computational biology, animal modeling, and clinical patient profiles to characterize the effect of aberrant splicing of a brain-enriched alternative exon in the membrane-binding tumor suppressor Annexin A7 (ANXA7) on oncogene regulation and brain tumorigenesis. RESULTS: We show that aberrant splicing of a tissue-specific cassette exon in ANXA7 diminishes endosomal targeting and consequent termination of the signal of the EGFR oncoprotein during brain tumorigenesis. Splicing of this exon is mediated by the ribonucleoprotein Polypyrimidine Tract-Binding Protein 1 (PTBP1), which is normally repressed during brain development but, we find, is excessively expressed in glioblastomas through either gene amplification or loss of a neuron-specific microRNA, miR-124. Silencing of PTBP1 attenuates both malignancy and angiogenesis in a stem cell-derived glioblastoma animal model characterized by a high native propensity to generate tumor endothelium or vascular pericytes to support tumor growth. We show that EGFR amplification and PTBP1 overexpression portend a similarly poor clinical outcome, further highlighting the importance of PTBP1-mediated activation of EGFR

  19. FANCM c.5791C>T nonsense mutation (rs144567652) induces exon skipping, affects DNA repair activity and is a familial breast cancer risk factor.

    PubMed

    Peterlongo, Paolo; Catucci, Irene; Colombo, Mara; Caleca, Laura; Mucaki, Eliseos; Bogliolo, Massimo; Marin, Maria; Damiola, Francesca; Bernard, Loris; Pensotti, Valeria; Volorio, Sara; Dall'Olio, Valentina; Meindl, Alfons; Bartram, Claus; Sutter, Christian; Surowy, Harald; Sornin, Valérie; Dondon, Marie-Gabrielle; Eon-Marchais, Séverine; Stoppa-Lyonnet, Dominique; Andrieu, Nadine; Sinilnikova, Olga M; Mitchell, Gillian; James, Paul A; Thompson, Ella; Marchetti, Marina; Verzeroli, Cristina; Tartari, Carmen; Capone, Gabriele Lorenzo; Putignano, Anna Laura; Genuardi, Maurizio; Medici, Veronica; Marchi, Isabella; Federico, Massimo; Tognazzo, Silvia; Matricardi, Laura; Agata, Simona; Dolcetti, Riccardo; Della Puppa, Lara; Cini, Giulia; Gismondi, Viviana; Viassolo, Valeria; Perfumo, Chiara; Mencarelli, Maria Antonietta; Baldassarri, Margherita; Peissel, Bernard; Roversi, Gaia; Silvestri, Valentina; Rizzolo, Piera; Spina, Francesca; Vivanet, Caterina; Tibiletti, Maria Grazia; Caligo, Maria Adelaide; Gambino, Gaetana; Tommasi, Stefania; Pilato, Brunella; Tondini, Carlo; Corna, Chiara; Bonanni, Bernardo; Barile, Monica; Osorio, Ana; Benitez, Javier; Balestrino, Luisa; Ottini, Laura; Manoukian, Siranoush; Pierotti, Marco A; Renieri, Alessandra; Varesco, Liliana; Couch, Fergus J; Wang, Xianshu; Devilee, Peter; Hilbers, Florentine S; van Asperen, Christi J; Viel, Alessandra; Montagna, Marco; Cortesi, Laura; Diez, Orland; Balmaña, Judith; Hauke, Jan; Schmutzler, Rita K; Papi, Laura; Pujana, Miguel Angel; Lázaro, Conxi; Falanga, Anna; Offit, Kenneth; Vijai, Joseph; Campbell, Ian; Burwinkel, Barbara; Kvist, Anders; Ehrencrona, Hans; Mazoyer, Sylvie; Pizzamiglio, Sara; Verderio, Paolo; Surralles, Jordi; Rogan, Peter K; Radice, Paolo

    2015-09-15

    Numerous genetic factors that influence breast cancer risk are known. However, approximately two-thirds of the overall familial risk remain unexplained. To determine whether some of the missing heritability is due to rare variants conferring high to moderate risk, we tested for an association between the c.5791C>T nonsense mutation (p.Arg1931*; rs144567652) in exon 22 of FANCM gene and breast cancer. An analysis of genotyping data from 8635 familial breast cancer cases and 6625 controls from different countries yielded an association between the c.5791C>T mutation and breast cancer risk [odds ratio (OR) = 3.93 (95% confidence interval (CI) = 1.28-12.11; P = 0.017)]. Moreover, we performed two meta-analyses of studies from countries with carriers in both cases and controls and of all available data. These analyses showed breast cancer associations with OR = 3.67 (95% CI = 1.04-12.87; P = 0.043) and OR = 3.33 (95% CI = 1.09-13.62; P = 0.032), respectively. Based on information theory-based prediction, we established that the mutation caused an out-of-frame deletion of exon 22, due to the creation of a binding site for the pre-mRNA processing protein hnRNP A1. Furthermore, genetic complementation analyses showed that the mutation influenced the DNA repair activity of the FANCM protein. In summary, we provide evidence for the first time showing that the common p.Arg1931* loss-of-function variant in FANCM is a risk factor for familial breast cancer. PMID:26130695

  20. The first exon of the c-myc proto-oncogene contains a novel positive control element.

    PubMed Central

    Yang, J Q; Remmers, E F; Marcu, K B

    1986-01-01

    We have identified a positive modulator within the c-myc first exon downstream of the gene's transcription initiation sites, P1 and P2. We introduced myc-CAT (chloramphenicol acetyltransferase) hybrid genes into three cell lines (BJAB, COS and HeLa) and measured their expression by either CAT enzymatic activity, S1 nuclease protection or by a nuclear 'run-on' transcription assay. Removal of 46 bp from the 3' end of the first exon results in a decrease of myc-CAT expression and P2 activity. A 438-bp exon 1 segment, lacking the normal myc promoters, efficiently drives the expression of SV40 early promoters. We find that this first exon segment efficiently functions as a positive modulator only in its sense orientation, 3' of a nearby promoter. The positive effects of the myc first exon and the SV40 enhancer are complementary. Images Fig. 2. Fig. 4. Fig. 5. Fig. 6. Fig. 7. Fig. 8. PMID:3030732

  1. RNA Folding Affects the Recruitment of SR Proteins by Mouse and Human Polypurinic Enhancer Elements in the Fibronectin EDA Exon

    PubMed Central

    Buratti, Emanuele; Muro, Andrés F.; Giombi, Maurizio; Gherbassi, Daniel; Iaconcig, Alessandra; Baralle, Francisco E.

    2004-01-01

    In humans, inclusion or exclusion of the fibronectin EDA exon is mainly regulated by a polypurinic enhancer element (exonic splicing enhancer [ESE]) and a nearby silencer element (exonic splicing silencer [ESS]). While human and mouse ESEs behave identically, mutations introduced into the homologous mouse ESS sequence result either in no change in splicing efficiency or in complete exclusion of the exon. Here, we show that this apparently contradictory behavior cannot be simply accounted for by a localized sequence variation between the two species. Rather, the nucleotide differences as a whole determine several changes in the respective RNA secondary structures. By comparing how the two different structures respond to homologous deletions in their putative ESS sequences, we show that changes in splicing behavior can be accounted for by a differential ESE display in the two RNAs. This is confirmed by RNA-protein interaction analysis of levels of SR protein binding to each exon. The immunoprecipitation patterns show the presence of complex multi-SR protein-RNA interactions that are lost with secondary-structure variations after the introduction of ESE and ESS variations. Taken together, our results demonstrate that the sequence context, in addition to the primary sequence identity, can heavily contribute to the making of functional units capable of influencing pre-mRNA splicing. PMID:14729981

  2. Transcriptome-based exon capture enables highly cost-effective comparative genomic data collection at moderate evolutionary scales

    PubMed Central

    2012-01-01

    Background To date, exon capture has largely been restricted to species with fully sequenced genomes, which has precluded its application to lineages that lack high quality genomic resources. We developed a novel strategy for designing array-based exon capture in chipmunks (Tamias) based on de novo transcriptome assemblies. We evaluated the performance of our approach across specimens from four chipmunk species. Results We selectively targeted 11,975 exons (~4 Mb) on custom capture arrays, and enriched over 99% of the targets in all libraries. The percentage of aligned reads was highly consistent (24.4-29.1%) across all specimens, including in multiplexing up to 20 barcoded individuals on a single array. Base coverage among specimens and within targets in each species library was uniform, and the performance of targets among independent exon captures was highly reproducible. There was no decrease in coverage among chipmunk species, which showed up to 1.5% sequence divergence in coding regions. We did observe a decline in capture performance of a subset of targets designed from a much more divergent ground squirrel genome (30 My), however, over 90% of the targets were also recovered. Final assemblies yielded over ten thousand orthologous loci (~3.6 Mb) with thousands of fixed and polymorphic SNPs among species identified. Conclusions Our study demonstrates the potential of a transcriptome-enabled, multiplexed, exon capture method to create thousands of informative markers for population genomic and phylogenetic studies in non-model species across the tree of life. PMID:22900609

  3. Bottom-up design of small molecules that stimulate exon 10 skipping in mutant MAPT pre-mRNA.

    PubMed

    Luo, Yiling; Disney, Matthew D

    2014-09-22

    One challenge in chemical biology is to develop small molecules that control cellular protein content. The amount and identity of proteins are influenced by the RNAs that encode them; thus, protein content in a cell could be affected by targeting mRNA. However, RNA has been traditionally difficult to target with small molecules. In this report, we describe controlling the protein products of the mutated microtubule-associated protein tau (MAPT) mature mRNA with a small molecule. MAPT mutations in exon 10 are associated with inherited frontotemporal dementia and Parkinsonism linked to chromosome 17 (FTDP-17), an incurable disease that is directly caused by increased inclusion of exon 10 in MAPT mRNA. Recent studies have shown that mutations within a hairpin at the MAPT exon 10-intron junction decrease the thermodynamic stability of the RNA, increasing binding to U1 snRNP and thus exon 10 inclusion. Therefore, we designed small molecules that bind and stabilize a mutant MAPT by using Inforna, a computational approach based on information about RNA-small-molecule interactions. The optimal compound selectively bound the mutant MAPT hairpin and thermodynamically stabilized its folding, facilitating exon 10 exclusion. PMID:25115866

  4. Proteomic profiling of antisense-induced exon skipping reveals reversal of pathobiochemical abnormalities in dystrophic mdx diaphragm

    PubMed Central

    Doran, Philip; Wilton, Steve D.; Fletcher, Sue; Ohlendieck, Kay

    2009-01-01

    The disintegration of the dystrophin-glycoprotein complex represents the initial pathobiochemical insult in Duchenne muscular dystrophy. However, secondary changes in signalling, energy metabolism and ion homeostasis are probably the main factors that eventually cause progressive muscle wasting. Thus, for the proper evaluation of novel therapeutic approaches, it is essential to analyse the reversal of both primary and secondary abnormalities in treated muscles. Antisense oligomer-mediated exon skipping promises functional restoration of the primary deficiency in dystrophin. In this study, an established phosphorodiamidate morpholino oligomer coupled to a cell-penetrating peptide was employed for the specific removal of exon 23 in the mutated mouse dystrophin gene transcript. Using DIGE analysis, we could show the reversal of secondary pathobiochemical abnormalities in the dystrophic diaphragm following exon-23 skipping. In analogy to the restoration of dystrophin, β-dystroglycan and neuronal nitric oxide synthase, the muscular dystrophy-associated differential expression of calsequestrin, adenylate kinase, aldolase, mitochondrial creatine kinase and cvHsp was reversed in treated muscle fibres. Hence, the re-establishment of Dp427 coded by the transcript missing exon 23 has counter-acted dystrophic alterations in Ca2+-handling, nucleotide metabolism, bioenergetic pathways and cellular stress response. This clearly establishes the exon-skipping approach as a realistic treatment strategy for diminishing diverse downstream alterations in dystrophinopathy. PMID:19132684

  5. Screening exons 16 and 17 of the amyloid precursor protein gene in sporadic early-onset Alzheimer's disease.

    PubMed

    Barber, Imelda S; García-Cárdenas, Jennyfer M; Sakdapanichkul, Chidchanok; Deacon, Christopher; Zapata Erazo, Gabriela; Guerreiro, Rita; Bras, Jose; Hernandez, Dena; Singleton, Andrew; Guetta-Baranes, Tamar; Braae, Anne; Clement, Naomi; Patel, Tulsi; Brookes, Keeley; Medway, Christopher; Chappell, Sally; Mann, David M; Morgan, Kevin

    2016-03-01

    Early-onset Alzheimer's disease (EOAD) can be familial (FAD) or sporadic EOAD (sEOAD); both have a disease onset ≤65 years of age. A total of 451 sEOAD samples were screened for known causative mutations in exons 16 and 17 of the amyloid precursor protein (APP) gene. Four samples were shown to be heterozygous for 1 of 3 known causative mutations: p.A713T, p.V717I, and p.V717G; this highlights the importance of screening EOAD patients for causative mutations. Additionally, we document an intronic 6 base pair (bp) deletion located 83 bp downstream of exon 17 (rs367709245, IVS17 83-88delAAGTAT), which has a nonsignificantly increased minor allele frequency in our sEOAD cohort (0.006) compared to LOAD (0.002) and controls (0.002). To assess the effect of the 6-bp deletion on splicing, COS-7 and BE(2)-C cells were transfected with a minigene vector encompassing exon 17. There was no change in splicing of exon 17 from constructs containing either wild type or deletion inserts. Sequencing of cDNA generated from cerebellum and temporal cortex of a patient harboring the deletion found no evidence of transcripts with exon 17 removed. PMID:26803359

  6. Exon organization of the mouse entactin gene corresponds to the structural domains of the polypeptide and has regional homology to the low-density lipoprotein receptor gene

    SciTech Connect

    Durkin, M.E.; Chung, A.E.; Wewer, U.M.

    1995-03-20

    Entactin is a widespread basement membrane protein of 150 kDa that binds to type IV collagen and laminin. The complete exon-intron structure of the mouse entactin gene has been determined from {lambda} genomic DNA clones. The gene spans at least 65 kb and contains 20 exons. The exon organization of the mouse entactin gene closely corresponds to the organization of the polypeptide into distinct structural and functional domains. The two amino-terminal globular domains are encoded by three exons each. Single exons encode the two protease-sensitive, O-glycosylated linking regions. The six EGF-like repeats and the single thyroglobulin-type repeat are each encoded by separate exons. The carboxyl-terminal half of entactin displays sequence homology to the growth factor-like region of the low-density lipoprotein receptor, and in both genes this region is encoded by eight exons. The positions of four introns are also conserved in the homologous region of the two genes. These observations suggest that the entactin gene has evolved via exon shuffling. Finally, several sequence polymorphisms useful for gene linkage analysis were found in the 3{prime} noncoding region of the last exon. 52 refs., 8 figs.

  7. The Drosophila melanogaster multidrug-resistance protein 1 (MRP1) homolog has a novel gene structure containing two variable internal exons.

    PubMed

    Grailles, Marine; Brey, Paul T; Roth, Charles W

    2003-03-27

    Drosophila melanogaster has a gene very similar to human MRP1 that encodes a full ABC-transporter containing three membrane-spanning domains and two nucleotide-binding domains. This 19 exon insect gene, dMRP (FBgn0032456), spans slightly more than 22 kb. The cDNA SD07655 representing this gene was sequenced and found to contain sequences from 12 exons including single copies of two exons having multiple genomic copies. The gene contains two variant copies of exon 4 and seven of exon 8. While a cDNA contains only one version of each variable exon, all forms of these variable exons were detected in adult fly mRNA. These results predict that Drosophila could make 14 different MRP isoforms from a single gene by substituting different variable exons. This is the first report of any organism using differential splicing of alternative, internal exons, to produce such a large array of MRP isoforms having the same size, but with limited and defined internal variations. Defining the functional differences in the dMRP isoforms should provide clues to the structure/function relationships of the amino acids in these MRP domains, both for the insect enzyme and for those of other species. PMID:12706887

  8. In vivo role of the HNF4α AF-1 activation domain revealed by exon swapping

    PubMed Central

    Briançon, Nadège; Weiss, Mary C

    2006-01-01

    The gene encoding the nuclear receptor hepatocyte nuclear factor 4α (HNF4α) generates isoforms HNF4α1 and HNF4α7 from usage of alternative promoters. In particular, HNF4α7 is expressed in the pancreas whereas HNF4α1 is found in liver, and mutations affecting HNF4α function cause impaired insulin secretion and/or hepatic defects in humans and in tissue-specific ‘knockout' mice. HNF4α1 and α7 isoforms differ exclusively by amino acids encoded by the first exon which, in HNF4α1 but not in HNF4α7, includes the activating function (AF)-1 transactivation domain. To investigate the roles of HNF4α1 and HNF4α7 in vivo, we generated mice expressing only one isoform under control of both promoters, via reciprocal swapping of the isoform-specific first exons. Unlike Hnf4α gene disruption which causes embryonic lethality, these ‘α7-only' and ‘α1-only' mice are viable, indicating functional redundancy of the isoforms. However, the former show dyslipidemia and preliminary results indicate impaired glucose tolerance for the latter, revealing functional specificities of the isoforms. These ‘knock-in' mice provide the first test in vivo of the HNF4α AF-1 function and have permitted identification of AF-1-dependent target genes. PMID:16498401

  9. SinEx DB: a database for single exon coding sequences in mammalian genomes

    PubMed Central

    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 PMID:27278816

  10. Intron-exon organization of the gene for the multifunctional animal fatty acid synthase.

    PubMed Central

    Amy, C M; Williams-Ahlf, B; Naggert, J; Smith, S

    1992-01-01

    The complete intron-exon organization of the gene encoding a multifunctional mammalian fatty acid synthase has been elucidated, and specific exons have been assigned to coding sequences for the component domains of the protein. The rat gene is interrupted by 42 introns and the sequences bordering the splice-site junctions universally follow the GT/AG rule. However, of the 41 introns that interrupt the coding region of the gene, 23 split the reading frame in phase I, 14 split the reading frame in phase 0, and only 4 split the reading frame in phase II. Remarkably, 46% of the introns interrupt codons for glycine. With only one exception, boundaries between the constituent enzymes of the multifunctional polypeptide coincide with the location of introns in the gene. The significance of the predominance of phase I introns, the almost uniformly short length of the 42 introns and the overall small size of the gene, is discussed in relation to the evolution of multifunctional proteins. Images PMID:1736293

  11. Novel alleles of the transforming growth factor β-1 regulatory region and exon 1.

    PubMed

    Arrieta-Bolaños, E; Madrigal, J A; Shaw, B E

    2015-06-01

    Transforming growth factor β-1, encoded by the TGFB1 gene, is a cytokine that plays a central role in many physiologic and pathogenic processes with pleiotropic effects. Regulatory activity for this gene has been shown for 3.0 kb between positions -2665 and +423 from its translational start site. At least 17 TGFB1 regulatory region and exon 1 alleles have been defined on the basis of 18 polymorphic sites. Polymorphisms in TGFB1's regulatory region have been associated with differential levels of expression of this cytokine and to genetic risk in cancer and transplantation. In this report, we present 19 novel TGFB1 regulatory region and exon 1 alleles: p018-p036. Amplification of TGFB1's regulatory region was performed with an in-house protocol, and novel alleles were defined by either allele-specific amplification and/or molecular cloning of the amplicons, followed by sequencing in isolation. Three of these novel alleles (p018, p019, and p020) are shown to be formed by novel combinations of the aforementioned known polymorphic positions. Another 16 novel alleles are shown to carry additional known and unknown single-nucleotide polymorphisms. Polymorphism in TGFB1's regulatory region could have an impact on important processes, including embryogenesis, hematopoiesis, carcinogenesis, angiogenesis, fibrosis, immune responses, and transplantation, making its characterization necessary. PMID:25808355

  12. α-Synuclein and huntingtin exon 1 amyloid fibrils bind laterally to the cellular membrane

    PubMed Central

    Monsellier, Elodie; Bousset, Luc; Melki, Ronald

    2016-01-01

    Fibrillar aggregates involved in neurodegenerative diseases have the ability to spread from one cell to another in a prion-like manner. The underlying molecular mechanisms, in particular the binding mode of the fibrils to cell membranes, are poorly understood. In this work we decipher the modality by which aggregates bind to the cellular membrane, one of the obligatory steps of the propagation cycle. By characterizing the binding properties of aggregates made of α-synuclein or huntingtin exon 1 protein displaying similar composition and structure but different lengths to mammalian cells we demonstrate that in both cases aggregates bind laterally to the cellular membrane, with aggregates extremities displaying little or no role in membrane binding. Lateral binding to artificial liposomes was also observed by transmission electron microscopy. In addition we show that although α-synuclein and huntingtin exon 1 fibrils bind both laterally to the cellular membrane, their mechanisms of interaction differ. Our findings have important implications for the development of future therapeutic tools that aim to block protein aggregates propagation in the brain. PMID:26757959

  13. The exon junction complex is required for definition and excision of neighboring introns in Drosophila

    PubMed Central

    Hayashi, Rippei; Handler, Dominik; Ish-Horowicz, David

    2014-01-01

    Splicing of pre-mRNAs results in the deposition of the exon junction complex (EJC) upstream of exon–exon boundaries. The EJC plays crucial post-splicing roles in export, translation, localization, and nonsense-mediated decay of mRNAs. It also aids faithful splicing of pre-mRNAs containing large introns, albeit via an unknown mechanism. Here, we show that the core EJC plus the accessory factors RnpS1 and Acinus aid in definition and efficient splicing of neighboring introns. This requires prior deposition of the EJC in close proximity to either an upstream or downstream splicing event. If present in isolation, EJC-dependent introns are splicing-defective also in wild-type cells. Interestingly, the most affected intron belongs to the piwi locus, which explains the reported transposon desilencing in EJC-depleted Drosophila ovaries. Based on a transcriptome-wide analysis, we propose that the dependency of splicing on the EJC is exploited as a means to control the temporal order of splicing events. PMID:25081352

  14. The exon junction complex controls transposable element activity by ensuring faithful splicing of the piwi transcript

    PubMed Central

    Malone, Colin D.; Mestdagh, Claire; Akhtar, Junaid; Kreim, Nastasja; Deinhard, Pia; Sachidanandam, Ravi; Treisman, Jessica

    2014-01-01

    The exon junction complex (EJC) is a highly conserved ribonucleoprotein complex that binds RNAs during splicing and remains associated with them following export to the cytoplasm. While the role of this complex in mRNA localization, translation, and degradation has been well characterized, its mechanism of action in splicing a subset of Drosophila and human transcripts remains to be elucidated. Here, we describe a novel function for the EJC and its splicing subunit, RnpS1, in preventing transposon accumulation in both Drosophila germline and surrounding somatic follicle cells. This function is mediated specifically through the control of piwi transcript splicing, where, in the absence of RnpS1, the fourth intron of piwi is retained. This intron contains a weak polypyrimidine tract that is sufficient to confer dependence on RnpS1. Finally, we demonstrate that RnpS1-dependent removal of this intron requires splicing of the flanking introns, suggesting a model in which the EJC facilitates the splicing of weak introns following its initial deposition at adjacent exon junctions. These data demonstrate a novel role for the EJC in regulating piwi intron excision and provide a mechanism for its function during splicing. PMID:25104425

  15. A novel whole exon deletion in WWOX gene causes early epilepsy, intellectual disability and optic atrophy.

    PubMed

    Ben-Salem, Salma; Al-Shamsi, Aisha M; John, Anne; Ali, Bassam R; Al-Gazali, Lihadh

    2015-05-01

    Recent studies have implicated the WW domain-containing oxidoreductase encoding gene (WWOX) in a severe form of autosomal recessive neurological disorder. This condition showed an overlapping spectrum of clinical features including spinocerebellar ataxia associated with generalized seizures and delayed psychomotor development to growth retardation, spasticity, and microcephaly. We evaluated a child from a consanguineous Emirati family that presented at birth with growth retardation, microcephaly, epileptic seizures, and later developed spasticity and delayed psychomotor development. Screening for deletions and duplications using whole-chromosomal microarray analysis identified a novel homozygous microdeletion encompassing exon 5 of the WWOX gene. Analysis of parental DNA indicated that this deletion was inherited from both parents and lies within a large region of homozygosity. Sanger sequencing of the cDNA showed that the deletion resulted in exon 5 skipping leading to a frame-shift and creating a premature stop codon at amino acid position 212. Quantification of mRNA revealed striking low level of WWOX expression in the child and moderate level of expression in the mother compared to a healthy control. To the best of our knowledge, this is the first homozygous germline structural variation in WWOX gene resulting in truncated transcripts that were presumably subject to NMD pathway. Our findings extend the clinical and genetic spectrum of WWOX mutations and support a crucial role of this gene in neurological development. PMID:25403906

  16. Mitochondrial intronic open reading frames in Podospora: Mobility and consecutive exonic sequence variations

    SciTech Connect

    Sellem, C.H.; Rossignol, M.; Belcour, L.

    1996-06-01

    The mitochondrial genome of 23 wild-type strains belonging to three different species of the filamentous fungus Podospora was examined. Among the 15 optical sequences identified are two intronic reading frames, nad1-i4-orf1 and cox1-i7-orf2. We show that the presence of these sequences was strictly correlated with tightly clustered nucleotide substitutions in the adjacent exon. This correlation applies to the presence or absence of closely related open reading frames (ORFs), found at the same genetic locations, in all the Pyrenomycete genera examined. The recent gain of these optional ORFs in the evolution of the genus Podospora probably account for such sequence differences. In the homoplasmic progeny from heteroplasmons constructed between Podospora strains differing by the presence of these optional ORFs, nad1-i4-orf1 and cox1-i7-orf2 appeared highly invasive. Sequence comparisons in the nad1-i4 intron of various strains of the Pyrenomycete family led us to propose a scenario of its evolution that includes several events of loss and gain of intronic ORFs. These results strongly reinforce the idea that group I intronic ORFs are mobile elements and that their transfer, and comcomitant modification of the adjacent exon, could participate in the modular evolution of mitochondrial genomes. 46 refs., 5 figs., 2 tabs.

  17. SinEx DB: a database for single exon coding sequences in mammalian genomes.

    PubMed

    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. PMID:27278816

  18. Coexisting JAK2V617F and CALR Exon 9 Mutation in Essential Thrombocythemia.

    PubMed

    Rashid, Munazza; Ahmed, Rifat Zubair; Ahmed, Shariq; Nadeem, Muhammad; Ahmed, Nuzhat; Shamsi, Tahir Sultan

    2016-06-01

    Classic "BCR-ABL1-negative" MPN is an operational sub-category of MPN that includes polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF) harboring JAK2V617F as the most common mutation. JAK2V617F can be detected in about 95 % of patients with PV while remaining 5 % of PV patients carry a somatic mutation of JAK2 exon 12. Approximately one-third of patients with ET or PMF do not carry any mutation in JAK2 or MPL. In December 2013, mutations were described in calreticulin (CALR) gene in 67-71 and 56-88 % of JAK2V617F and MPL negative patients with ET and PMF, respectively. Since this discovery CALR mutations have been reported to be mutually exclusive with JAK2V617F or MPL mutations. However recently few studies (eleven published reports) reported the coexistence of JAK2V617F and CALR in MPN. In the present study we are reporting JAK2V617F positive ET patient from our center with coexisting CALR exon 9 mutation type c.1214_1225del12 (p.E405_D408del) that was never reported before as a coexisting mutation and describing in detail the clinical outcomes. PMID:27408370

  19. The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina

    PubMed Central

    Murphy, Daniel; Carstens, Russ

    2016-01-01

    Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such “switch-like” exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments. PMID:27541351

  20. The thermodynamic patterns of eukaryotic genes suggest a mechanism for intron-exon recognition.

    PubMed

    Nedelcheva-Veleva, Marina N; Sarov, Mihail; Yanakiev, Ivan; Mihailovska, Eva; Ivanov, Miroslav P; Panova, Greta C; Stoynov, Stoyno S

    2013-01-01

    The essential cis- and trans-acting elements required for RNA splicing have been defined, however, the detailed molecular mechanisms underlying intron-exon recognition are still unclear. Here we demonstrate that the ratio between stability of mRNA/DNA and DNA/DNA duplexes near 3'-spice sites is a characteristic feature that can contribute to intron-exon differentiation. Remarkably, throughout all transcripts, the most unstable mRNA/DNA duplexes, compared with the corresponding DNA/DNA duplexes, are situated upstream of the 3'-splice sites and include the polypyrimidine tracts. This characteristic instability is less pronounced in weak alternative splice sites and disease-associated cryptic 3'-splice sites. Our results suggest that this thermodynamic pattern can prevent the re-annealing of mRNA to the DNA template behind the RNA polymerase to ensure access of the splicing machinery to the polypyrimidine tract and the branch point. In support of this mechanism, we demonstrate that RNA/DNA duplex formation at this region prevents pre-spliceosome A complex assembly. PMID:23817463

  1. Exon skipping and translation in patients with frameshift deletions in the dystrophin gene

    SciTech Connect

    Sherratt, T.G.; Dubowitz, V.; Sewry, C.A.; Strong, P.N. ); Vulliamy, T. )

    1993-11-01

    Although many Duchenne muscular dystrophy patients have a deletion in the dystrophin gene which disrupts the translational reading frame, they express dystrophin in a small proportion of skeletal muscle fibers ([open quotes]revertant fibers[close quotes]). Antibody studies have shown, indirectly, that dystrophin synthesis in revertant fibers is facilitated by a frame-restoring mechanism; in the present study, the feasibility of mRNA splicing was investigated. Dystrophin transcripts were analyzed in skeletal muscle from individuals possessing revertant fibers and a frameshift deletion in the dystrophin gene. In each case a minor in-frame transcript was detected, in which exons adjacent to those deleted from the genome had been skipped. There appeared to be some correlation between the levels of in-frame transcripts and the predicted translation products. Low levels of alternatively spliced transcripts were also present in normal muscle. The results provide further evidence of exon skipping in the dystrophin gene and indicate that this may be involved in the synthesis of dystrophin by revertant fibers. 44 refs., 12 figs.

  2. Modeling Exon-Specific Bias Distribution Improves the Analysis of RNA-Seq Data.

    PubMed

    Liu, Xuejun; Zhang, Li; Chen, Songcan

    2015-01-01

    RNA-seq technology has become an important tool for quantifying the gene and transcript expression in transcriptome study. The two major difficulties for the gene and transcript expression quantification are the read mapping ambiguity and the overdispersion of the read distribution along reference sequence. Many approaches have been proposed to deal with these difficulties. A number of existing methods use Poisson distribution to model the read counts and this easily splits the counts into the contributions from multiple transcripts. Meanwhile, various solutions were put forward to account for the overdispersion in the Poisson models. By checking the similarities among the variation patterns of read counts for individual genes, we found that the count variation is exon-specific and has the conserved pattern across the samples for each individual gene. We introduce Gamma-distributed latent variables to model the read sequencing preference for each exon. These variables are embedded to the rate parameter of a Poisson model to account for the overdispersion of read distribution. The model is tractable since the Gamma priors can be integrated out in the maximum likelihood estimation. We evaluate the proposed approach, PGseq, using four real datasets and one simulated dataset, and compare its performance with other popular methods. Results show that PGseq presents competitive performance compared to other alternatives in terms of accuracy in the gene and transcript expression calculation and in the downstream differential expression analysis. Especially, we show the advantage of our method in the analysis of low expression. PMID:26448625

  3. The Musashi 1 Controls the Splicing of Photoreceptor-Specific Exons in the Vertebrate Retina.

    PubMed

    Murphy, Daniel; Cieply, Benjamin; Carstens, Russ; Ramamurthy, Visvanathan; Stoilov, Peter

    2016-08-01

    Alternative pre-mRNA splicing expands the coding capacity of eukaryotic genomes, potentially enabling a limited number of genes to govern the development of complex anatomical structures. Alternative splicing is particularly prevalent in the vertebrate nervous system, where it is required for neuronal development and function. Here, we show that photoreceptor cells, a type of sensory neuron, express a characteristic splicing program that affects a broad set of transcripts and is initiated prior to the development of the light sensing outer segments. Surprisingly, photoreceptors lack prototypical neuronal splicing factors and their splicing profile is driven to a significant degree by the Musashi 1 (MSI1) protein. A striking feature of the photoreceptor splicing program are exons that display a "switch-like" pattern of high inclusion levels in photoreceptors and near complete exclusion outside of the retina. Several ubiquitously expressed genes that are involved in the biogenesis and function of primary cilia produce highly photoreceptor specific isoforms through use of such "switch-like" exons. Our results suggest a potential role for alternative splicing in the development of photoreceptors and the conversion of their primary cilia to the light sensing outer segments. PMID:27541351

  4. Acetylation within the First 17 Residues of Huntingtin Exon 1 Alters Aggregation and Lipid Binding.

    PubMed

    Chaibva, Maxmore; Jawahery, Sudi; Pilkington, Albert W; Arndt, James R; Sarver, Olivia; Valentine, Stephen; Matysiak, Silvina; Legleiter, Justin

    2016-07-26

    Huntington's disease (HD) is a genetic neurodegenerative disorder caused by an expanded polyglutamine (polyQ) domain near the N-terminus of the huntingtin (htt) protein. Expanded polyQ leads to htt aggregation. The first 17 amino acids (Nt(17)) in htt comprise a lipid-binding domain that undergoes a number of posttranslational modifications that can modulate htt toxicity and subcellular localization. As there are three lysines within Nt(17), we evaluated the impact of lysine acetylation on htt aggregation in solution and on model lipid bilayers. Acetylation of htt-exon1(51Q) and synthetic truncated htt-exon 1 mimicking peptides (Nt(17)-Q35-P10-KK) was achieved using a selective covalent label, sulfo-N-hydroxysuccinimide (NHSA). With this treatment, all three lysine residues (K6, K9, and K15) in Nt(17) were significantly acetylated. N-terminal htt acetylation retarded fibril formation in solution and promoted the formation of larger globular aggregates. Acetylated htt also bound lipid membranes and disrupted the lipid bilayer morphology less aggressively compared with the wild-type. Computational studies provided mechanistic insights into how acetylation alters the interaction of Nt(17) with lipid membranes. Our results highlight that N-terminal acetylation influences the aggregation of htt and its interaction with lipid bilayers. PMID:27463137

  5. Modeling Exon-Specific Bias Distribution Improves the Analysis of RNA-Seq Data

    PubMed Central

    Liu, Xuejun; Zhang, Li; Chen, Songcan

    2015-01-01

    RNA-seq technology has become an important tool for quantifying the gene and transcript expression in transcriptome study. The two major difficulties for the gene and transcript expression quantification are the read mapping ambiguity and the overdispersion of the read distribution along reference sequence. Many approaches have been proposed to deal with these difficulties. A number of existing methods use Poisson distribution to model the read counts and this easily splits the counts into the contributions from multiple transcripts. Meanwhile, various solutions were put forward to account for the overdispersion in the Poisson models. By checking the similarities among the variation patterns of read counts for individual genes, we found that the count variation is exon-specific and has the conserved pattern across the samples for each individual gene. We introduce Gamma-distributed latent variables to model the read sequencing preference for each exon. These variables are embedded to the rate parameter of a Poisson model to account for the overdispersion of read distribution. The model is tractable since the Gamma priors can be integrated out in the maximum likelihood estimation. We evaluate the proposed approach, PGseq, using four real datasets and one simulated dataset, and compare its performance with other popular methods. Results show that PGseq presents competitive performance compared to other alternatives in terms of accuracy in the gene and transcript expression calculation and in the downstream differential expression analysis. Especially, we show the advantage of our method in the analysis of low expression. PMID:26448625

  6. Papilledema Due to Mirtazapine

    PubMed Central

    Ceylan, Mehmet Emin; Evrensel, Alper; Cömert, Gökçe

    2016-01-01

    Background: Mirtazapine is a tetracyclic antidepressant that enhances both noradrenergic and serotonergic transmission. The most common cause of papilledema is increased intracranial pressure due to brain tumor. Also it may occur as a result of idiopathic intracranial hypertension (IIH, pseudo tumor cerebri). Moreover, papilledema may also develop due to retinitis, vasculitis, Graves’ disease, hypertension, leukemia, lymphoma, diabetes mellitus and radiation. Case Report: In this article, a patient who developed papilledema while under treatment with mirtazapine (30 mg/day) for two years and recovered with termination of mirtazapine treatment was discussed to draw the attention of clinicians to this side effect of mirtazapine. Conclusion: Idiopathic intracranial hypertension and papilledema due to psychotropic drugs has been reported in the literature. Mirtazapine may rarely cause peripheral edema. However, papilledema due to mirtazapine has not been previously reported. Although papilledema is a very rare side effect of an antidepressant treatment, fundoscopic examinations of patients must be performed regularly. PMID:27308085

  7. A fusion protein of HCMV IE1 exon4 and IE2 exon5 stimulates potent cellular immunity in an MVA vaccine vector

    SciTech Connect

    Wang, Z.; Zhou, W.; Srivastava, T.; La Rosa, C.; Mandarino, A.; Forman, S.J.; Zaia, J.A.; Britt, W.J.; Diamond, D.J.

    2008-08-01

    A therapeutic CMV vaccine incorporating an antigenic repertoire capable of eliciting a cellular immune response has yet to be successfully implemented for patients who already have acquired an infection. To address this problem, we have developed a vaccine candidate derived from modified vaccinia Ankara (MVA) that expresses three immunodominant antigens (pp65, IE1, IE2) from CMV. The novelty of this vaccine is the fusion of two adjacent exons from the immediate-early region of CMV, their successful expression in MVA, and robust immunogenicity in both primary and memory response models. Evaluation of the immunogenicity of the viral vaccine in mouse models shows that it can stimulate primary immunity against all three antigens in both the CD4{sup +} and CD8{sup +} T cell subsets. Evaluation of human PBMC from healthy CMV-positive donors or patients within 6 months of receiving hematopoietic cell transplant shows robust stimulation of existing CMV-specific CD4{sup +} and CD8{sup +} T cell subsets.

  8. Exon 19 L747P mutation presented as a primary resistance to EGFR-TKI: a case report

    PubMed Central

    Wang, Yu-Ting; Ning, Wei-Wei; Li, Jing

    2016-01-01

    Active mutations of the EGFR gene have been proved to predict the activity of EGFR-TKI. The most common mutations are the exon 19 deletion and exon 21 point mutation, both of which are sensitive to EGFR-TKI. However, rare EGFR mutations or complex mutations still exist, and data of which are scarce and controversial. Their response to EGFR-TKI remains uncertain. We presented a patient diagnosed with stage IV lung adenocarcinoma who was found to have the EGFR mutation in exon 19 (L747P) before any treatment. The disease progressed 2 months after the chemotherapy containing cisplatin and pemetrexed, and erlotinib was administered, but there was no response found. This EGFR-TKI naïve patient failed to achieve the desired effect with the therapy of EGFR-TKI. L747P may be associated with primary resistance to EGFR-TKI in this case. PMID:27499993

  9. AON-mediated Exon Skipping Restores Ciliation in Fibroblasts Harboring the Common Leber Congenital Amaurosis CEP290 Mutation

    PubMed Central

    Gerard, Xavier; Perrault, Isabelle; Hanein, Sylvain; Silva, Eduardo; Bigot, Karine; Defoort-Delhemmes, Sabine; Rio, Marlèene; Munnich, Arnold; Scherman, Daniel; Kaplan, Josseline; Kichler, Antoine; Rozet, Jean-Michel

    2012-01-01

    Leber congenital amaurosis (LCA) is a severe hereditary retinal dystrophy responsible for congenital or early-onset blindness. The most common disease-causing mutation (>10%) is located deep in intron 26 of the CEP290 gene (c.2991+1655A>G). It creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. In the present study, we show that the use of antisense oligonucleotides (AONs) allow an efficient skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients. These data support the feasibility of an AON-mediated exon skipping strategy to correct the aberrant splicing. PMID:23344081

  10. Exonic Splicing Mutations Are More Prevalent than Currently Estimated and Can Be Predicted by Using In Silico Tools

    PubMed Central

    Soukarieh, Omar; Gaildrat, Pascaline; Hamieh, Mohamad; Drouet, Aurélie; Baert-Desurmont, Stéphanie; Frébourg, Thierry; Tosi, Mario; Martins, Alexandra

    2016-01-01

    The identification of a causal mutation is essential for molecular diagnosis and clinical management of many genetic disorders. However, even if next-generation exome sequencing has greatly improved the detection of nucleotide changes, the biological interpretation of most exonic variants remains challenging. Moreover, particular attention is typically given to protein-coding changes often neglecting the potential impact of exonic variants on RNA splicing. Here, we used the exon 10 of MLH1, a gene implicated in hereditary cancer, as a model system to assess the prevalence of RNA splicing mutations among all single-nucleotide variants identified in a given exon. We performed comprehensive minigene assays and analyzed patient’s RNA when available. Our study revealed a staggering number of splicing mutations in MLH1 exon 10 (77% of the 22 analyzed variants), including mutations directly affecting splice sites and, particularly, mutations altering potential splicing regulatory elements (ESRs). We then used this thoroughly characterized dataset, together with experimental data derived from previous studies on BRCA1, BRCA2, CFTR and NF1, to evaluate the predictive power of 3 in silico approaches recently described as promising tools for pinpointing ESR-mutations. Our results indicate that ΔtESRseq and ΔHZEI-based approaches not only discriminate which variants affect splicing, but also predict the direction and severity of the induced splicing defects. In contrast, the ΔΨ-based approach did not show a compelling predictive power. Our data indicates that exonic splicing mutations are more prevalent than currently appreciated and that they can now be predicted by using bioinformatics methods. These findings have implications for all genetically-caused diseases. PMID:26761715

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

    SciTech Connect

    Lindor, N.M.; Sobell, J.L.; Thibodeau, S.N.

    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.

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

    PubMed

    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

  13. The complexity of the sylvatic cycle of Trypanosoma cruzi in Rio de Janeiro state (Brazil) revealed by the non-transcribed spacer of the mini-exon gene.

    PubMed

    Fernandes, O; Mangia, R H; Lisboa, C V; Pinho, A P; Morel, C M; Zingales, B; Campbell, D A; Jansen, A M

    1999-02-01

    American trypanosamiasis occurs in nature as a sylvatic cycle, where Trypanosoma cruzi interacts with wild triatomines and mammalian reservoirs, such as marsupials, rodents, armadillos and other animals. Due to difficulties in trying to isolate T. cruzi stocks from the sylvatic cycle, very few studies have been performed in order to understand the parasite infection in natural environments. Traditionally T. cruzi has been considered to be composed of a highly heterogeneous population of parasites. In contrast, the mini-exon and the 24S alpha rRNA gene loci have shown that T. cruzi stocks can be clustered in 2 major phylogenetic groups: lineage 1 and lineage 2. In this report, 68 recently isolated T. cruzi samples from the sylvatic cycle belonging to different geographical areas in Rio de Janeiro, Brazil, have been typed based on a variable spot in the non-transcribed spacer of the mini-exon gene. Eight isolates were from triatomines, 26 stocks were from golden-lion tamarins, 31 from opossums, 2 from rodents and 1 from a three-toed sloth. Thirty (44%-30/68) isolates were typed as lineage 1, while 36 (53%-36/68) isolates were typed as lineage 2. Two opossums presented mixed infection. Therefore, 3% (2/68) of the isolates were typed as lineage 1 + lineage 2. Using these geographical regions as models of sylvatic environments, it was observed that 96% of the Didelphis marsupialis were infected by lineage 2 isolates, while all 26 golden-lion tamarins were infected by lineage 1. The results show preferential association of the 2 lineages of T. cruzi with different hosts, composing the complexity of the sylvatic cycle. PMID:10028530

  14. Hardy–Weinberg equilibrium analysis of the 48 bp VNTR in the III exon of the DRD4 gene in a sample of parents of ADHD cases

    PubMed Central

    Trejo, Salvador; Toscano-Flores, José J; Matute, Esmeralda; Ramírez-Dueñas, María de Lourdes

    2015-01-01

    The aim of this study was to obtain the genotype and gene frequency from parents of children with attention-deficit/hyperactivity disorder (ADHD) and then assess the Hardy–Weinberg equilibrium of genotype frequency of the variable number tandem repeat (VNTR) III exon of the dopamine receptor D4 (DRD4) gene. The genotypes of the III exon of 48 bp VNTR repeats of the DRD4 gene were determined by polymerase chain reaction in a sample of 30 parents of ADHD cases. In the 60 chromosomes analyzed, the following frequencies of DRD4 gene polymorphisms were observed: six chromosomes (c) with two repeat alleles (r) (10%); 1c with 3r (1.5%); 36c with 4r (60%); 1c with 5r (1.5%); and 16c with 7r (27%). The genotypic distribution of the 30 parents was two parents (p) with 2r/2r (6.67%); 1p with 2r/4r (3.33%); 1p with 2r/5r (3.33%); 1p with 3r/4r (3.33%); 15p with 4r/4r (50%); 4p with 4r/7r (13.33); and 6p with 7r/7r (20%). A Hardy–Weinberg disequilibrium (χ2=13.03, P<0.01) was found due to an over-representation of the 7r/7r genotype. These results suggest that the 7r polymorphism of the DRD4 gene is associated with the ADHD condition in a Mexican population. PMID:26082657

  15. Hardy-Weinberg equilibrium analysis of the 48 bp VNTR in the III exon of the DRD4 gene in a sample of parents of ADHD cases.

    PubMed

    Trejo, Salvador; Toscano-Flores, José J; Matute, Esmeralda; Ramírez-Dueñas, María de Lourdes

    2015-01-01

    The aim of this study was to obtain the genotype and gene frequency from parents of children with attention-deficit/hyperactivity disorder (ADHD) and then assess the Hardy-Weinberg equilibrium of genotype frequency of the variable number tandem repeat (VNTR) III exon of the dopamine receptor D4 (DRD4) gene. The genotypes of the III exon of 48 bp VNTR repeats of the DRD4 gene were determined by polymerase chain reaction in a sample of 30 parents of ADHD cases. In the 60 chromosomes analyzed, the following frequencies of DRD4 gene polymorphisms were observed: six chromosomes (c) with two repeat alleles (r) (10%); 1c with 3r (1.5%); 36c with 4r (60%); 1c with 5r (1.5%); and 16c with 7r (27%). The genotypic distribution of the 30 parents was two parents (p) with 2r/2r (6.67%); 1p with 2r/4r (3.33%); 1p with 2r/5r (3.33%); 1p with 3r/4r (3.33%); 15p with 4r/4r (50%); 4p with 4r/7r (13.33); and 6p with 7r/7r (20%). A Hardy-Weinberg disequilibrium (χ (2)=13.03, P<0.01) was found due to an over-representation of the 7r/7r genotype. These results suggest that the 7r polymorphism of the DRD4 gene is associated with the ADHD condition in a Mexican population. PMID:26082657

  16. Providers get their due.

    PubMed

    Morrissey, J

    1994-11-01

    Providers are getting their due, but only after employing computer software programs to help sort through the complex managed-care contracts they've negotiated. More and more accounting departments are relying on contract management systems to ensure accurate billing. PMID:10138187

  17. Paying Their Dues.

    ERIC Educational Resources Information Center

    Scalzo, Teresa

    1995-01-01

    Some colleges and universities have found that alumni prefer to have ownership of their alumni association, and such a membership program can raise revenues for the institution while providing a valuable communication tool. A strong dues program can work well with an annual giving campaign. A variety of membership structures is possible. Details…

  18. Mapping Human Pluripotent-to-Cardiomyocyte Differentiation: Methylomes, Transcriptomes, and Exon DNA Methylation "Memories".

    PubMed

    Tompkins, Joshua D; Jung, Marc; Chen, Chang-Yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A; Riggs, Arthur D

    2016-02-01

    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. PMID:26981572

  19. Detection of a novel mutation in exon 20 of the BRCA1 gene.

    PubMed

    Chakraborty, Abhijit; Katarkar, Atul; Chaudhuri, Keya; Mukhopadhyay, Ashis; Basak, Jayasri

    2013-12-01

    Hereditary breast cancer constitutes 5-10% of all breast cancer cases. Inherited mutations in the BRCA1 and BRCA2 tumor-suppressor genes account for the majority of hereditary breast cancer cases. The BRCA1 C-terminal region (BRCT) has a functional duplicated globular domain, which helps with DNA damage repair and cell cycle checkpoint protein control. More than 100 distinct BRCA1 missense variants with structural and functional effects have been documented within the BRCT domain. Interpreting the results of mutation screening of tumor-suppressor genes that can have high-risk susceptibility mutations is increasingly important in clinical practice. This study includes a novel mutation, p.His1746 Pro (c.5237A>C), which was found in BRCA1 exon 20 of a breast cancer patient. In silico analysis suggests that this mutation could alter the stability and orientation of the BRCT domain and the differential binding of the BACH1 substrate. PMID:24297685

  20. Dipole angular entropy techniques for intron-exon segregation in DNA

    NASA Astrophysics Data System (ADS)

    Subramanian, Nithya; Bose, R.

    2012-04-01

    We propose techniques for computing the angular entropies of DNA sequences, based on the orientations of the dipole moments of the nucleotide bases. The angles of the dipole moment vectors of the bases are used to compute the dipole angular entropy and the Fourier harmonics of the angles are used to compute the dipole angular spectral entropy for a given sequence. We also show that the coding (exons) and noncoding (introns) regions of the DNA can be segregated based on their dipole angular entropies and dipole angular spectral entropies. Segregation using these techniques is found to be computationally faster and more accurate than the previously reported methods. The proposed techniques can also be improvised to use the magnitude of the dipole moments of the bases in addition to the angles.

  1. Precursor protein of Alzheimer's disease A4 amyloid is encoded by 16 exons

    SciTech Connect

    Lemaire, H.G.; Kang, J.; Mueller-Hill, B. ); Salbaum, J.M.; Multhaup, G.; Beyreuther, K. ); Bayney, R.M.; Unterbeck, A. )

    1989-01-25

    Alzheimer's disease (AD) is characterized by the cerebral deposition of fibrillar aggregates of the amyloid A4 protein. Complementary DNA's coding for the precursor of the amyloid A4 protein have been described. In order to identify the structure of the precursor gene relevant clones from several human genomic libraries were isolated. Sequence analysis of the various clones revealed 16 exons to encode the 695 residue precursor protein (PreA4{sub 695}) of Alzheimer's disease amyloid A4 protein. The DNA sequence coding for the amyloid A4 protein is interrupted by an intron. This finding supports the idea that amyloid A4 protein arises by incomplete proteolysis of a larger precursor, and not by aberrant splicing.

  2. Progranulin mutation analysis: Identification of one novel mutation in exon 12 associated with frontotemporal dementia.

    PubMed

    Aswathy, Peethambaran Mallika; Jairani, Pushparajan Sulajamani; Raghavan, Sheela Kumari; Verghese, Joe; Gopala, Srinivas; Srinivas, Priya; Mathuranath, Pavagada Sivasankara

    2016-03-01

    Progranulin (PGRN) mutations account for an average of 15% of familial frontotemporal dementia (FTD) cases and 20% of total FTD cases worldwide. Here, we investigated the frequency of PGRN mutations in FTD patients (n = 116) from a clinical cohort of south India and detected one novel mutation located on exon 12 in a familial behavioral variant FTD patient (accounting for ∼1% of total FTD cases and 6% of familial FTD cases). This mutation was found to introduce a premature termination codon and the prematurely terminated messenger RNA may probably undergo nonsense-mediated decay. In enzyme-linked immunosorbent assay, the proband showed significantly reduced level of plasma PGRN (28 ng/mL) compared with controls (150 ± 38 ng/mL), which implicates haploinsufficiency as the pathogenic mechanism. PMID:26724960

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

    PubMed Central

    Tarpey, Patrick S; Smith, Raffaella; Pleasance, Erin; Whibley, Annabel; Edkins, Sarah; Hardy, Claire; O'Meara, Sarah; Latimer, Calli; Dicks, Ed; Menzies, Andrew; Stephens, Phil; Blow, Matt; Greenman, Chris; Xue, Yali; Tyler-Smith, Chris; Thompson, Deborah; Gray, Kristian; Andrews, Jenny; Barthorpe, Syd; Buck, Gemma; Cole, Jennifer; Dunmore, Rebecca; Jones, David; Maddison, Mark; Mironenko, Tatiana; Turner, Rachel; Turrell, Kelly; Varian, Jennifer; West, Sofie; Widaa, Sara; Wray, Paul; Teague, Jon; Butler, Adam; Jenkinson, Andrew; Jia, Mingming; Richardson, David; Shepherd, Rebecca; Wooster, Richard; Tejada, M Isabel; Martinez, Francisco; Carvill, Gemma; Goliath, Rene; de Brouwer, Arjan P M; van Bokhoven, Hans; Van Esch, Hilde; Chelly, Jamel; Raynaud, Martine; Ropers, Hans-Hilger; Abidi, Fatima E; Srivastava, Anand K; Cox, James; Luo, Ying; Mallya, Uma; Moon, Jenny; Parnau, Josef; Mohammed, Shehla; Tolmie, John L; Shoubridge, Cheryl; Corbett, Mark; Gardner, Alison; Haan, Eric; Rujirabanjerd, Sinitdhorn; Shaw, Marie; Vandeleur, Lucianne; Fullston, Tod; Easton, Douglas F; Boyle, Jackie; Partington, Michael; Hackett, Anna; Field, Michael; Skinner, Cindy; Stevenson, Roger E; Bobrow, Martin; Turner, Gillian; Schwartz, Charles E; Gecz, Jozef; Raymond, F Lucy; Futreal, P Andrew; Stratton, Michael R

    2010-01-01

    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), the largest direct screen for constitutional disease-causing mutations thus far reported. The screen has discovered nine genes implicated in XLMR, including SYP, ZNF711 and CASK reported here, confirming the power of this strategy. The study has, however, also highlighted issues confronting whole-genome sequencing screens, including the observation that loss of function of 1% or more of X-chromosome genes is compatible with apparently normal existence. PMID:19377476

  4. The exon junction complex as a node of post-transcriptional networks.

    PubMed

    Le Hir, Hervé; Saulière, Jérôme; Wang, Zhen

    2016-01-01

    The exon junction complex (EJC) is deposited onto mRNAs following splicing and adopts a unique structure, which can both stably bind to mRNAs and function as an anchor for diverse processing factors. Recent findings revealed that in addition to its established roles in nonsense-mediated mRNA decay, the EJC is involved in mRNA splicing, transport and translation. While structural studies have shed light on EJC assembly, transcriptome-wide analyses revealed differential EJC loading at spliced junctions. Thus, the EJC functions as a node of post-transcriptional gene expression networks, the importance of which is being revealed by the discovery of increasing numbers of EJC-related disorders. PMID:26670016

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

    PubMed Central

    Tompkins, Joshua D.; Jung, Marc; Chen, Chang-yi; Lin, Ziguang; Ye, Jingjing; Godatha, Swetha; Lizhar, Elizabeth; Wu, Xiwei; Hsu, David; Couture, Larry A.; Riggs, Arthur D.

    2016-01-01

    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. PMID:26981572

  6. Regions flanking exon 1 regulate constitutive expression of the human antithrombin gene.

    PubMed

    Fernandez-Rachubinski, F A; Weiner, J H; Blajchman, M A

    1996-11-15

    We have identified cis-acting elements and trans-acting factors that regulate constitutive expression of the human antithrombin gene. The activity of the sequences flanking the first exon of the gene was investigated using a luciferase-based reporter assay in transiently transfected HepG2, COS1, BSC40, and HeLa cells. Deletion analysis allowed the mapping of two elements able to promote antithrombin gene transcription in HepG2 and COS1 cells. The first element is located upstream of the first exon (-150/+68 nucleotides). The second element is in the first intervening sequence (+300/+700 nucleotides) and functions in an orientation opposite to that of the first. Footprint analysis showed three protected areas in the 5' upstream element at -92/-68 (element A), -14/+37 (element B), and -126/-100 nucleotides (element C). These elements acted as enhancers in luciferase reporter assays. Gel retardation analysis demonstrated that two liver-enriched transcription factors, hepatocyte nuclear factor 4 (HNF4) and CCAAT enhancer-binding protein (C/EBPa), bound to the 5' upstream element. HNF4 bound to elements A and C, whereas C/EBPa bound to element B. Element A also interacted with the ubiquitous nuclear hormone receptors chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1), thyroid hormone receptor alpha (TRalpha), peroxisome proliferator-activated receptor alpha(PPARalpha), and retinoid X receptor alpha (RXRalpha). In HepG2 and BSC40 cells, HNF4, C/EBPalpha, and RXRalpha activated luciferase expression from a reporter construct containing the 5'-upstream minimal antithrombin gene promoter, while COUP-TF1, TRalpha, and HNF3 (alpha or beta) repressed such expression. Our results show that constitutive expression of the human antithrombin gene depends in part upon the interplay of these transcription factors and suggest that signaling pathways regulated by these factors can modulate antithrombin gene transcription. PMID:8910619

  7. CD22 EXON 12 deletion as a pathogenic mechanism of human B-precursor leukemia

    PubMed Central

    Uckun, Fatih M.; Goodman, Patricia; Ma, Hong; Dibirdik, Ilker; Qazi, Sanjive

    2010-01-01

    Here, we report that primary leukemic cells from infants with newly diagnosed B-precursor leukemia express a truncated and functionally defective CD22 coreceptor protein that is unable to transmit apoptotic signals because it lacks most of the intracellular domain, including the key regulatory signal transduction elements and all of the cytoplasmic tyrosine residues. Expression of this structurally and functionally abnormal CD22 protein is associated with a very aggressive in vivo growth of patients’ primary leukemia cells causing disseminated overt leukemia in SCID mice. The abnormal CD22 coreceptor is encoded by a profoundly aberrant mRNA arising from a splicing defect that causes the deletion of exon 12 (c.2208-c.2327) (CD22ΔE12) and results in a truncating frameshift mutation. The splicing defect is associated with multiple homozygous mutations within a 132-bp segment of the intronic sequence between exons 12 and 13. These mutations cause marked changes in the predicted secondary structures of the mutant CD22 pre-mRNA sequences that affect the target motifs for the splicing factors hnRNP-L, PTB, and PCBP that are up-regulated in infant leukemia cells. Forced expression of the mutant CD22ΔE12 protein in transgenic mice perturbs B-cell development, as evidenced by B-precursor/B-cell hyperplasia, and corrupts the regulation of gene expression, causing reduced expression levels of several genes with a tumor suppressor function. We further show that CD22ΔE12-associated unique gene expression signature is a discriminating feature of newly diagnosed infant leukemia patients. These striking findings implicate CD22ΔE12 as a previously undescribed pathogenic mechanism in human B-precursor leukemia. PMID:20841423

  8. A deletion causing NF2 exon 9 skipping is associated with familial autosomal dominant intramedullary ependymoma

    PubMed Central

    Zemmoura, Ilyess; Vourc'h, Patrick; Paubel, Agathe; Parfait, Béatrice; Cohen, Joëlle; Bilan, Frédéric; Kitzis, Alain; Rousselot, Cécilia; Parker, Fabrice; François, Patrick; Andres, Christian R.

    2014-01-01

    Background Intramedullary ependymomas are rare and benign tumors in the adult. Little is known about their physiopathology, but the implication of the NF2 gene is suspected because of their presence in a third of patients with type 2 neurofibromatosis (NF2), a disorder caused by mutation of the NF2 gene. Methods We conducted a clinical and genetic study of a family in which 5 of 9 members suffered from intramedullary ependymoma. Karyotyping and CGH array analysis were performed on DNA from peripheral blood lymphocytes from affected participants. The NF2 gene sequences were then determined in DNA from 3 nonaffected and all 5 affected members of the family. Results Karyotype and CGH array findings were normal. Sequencing of NF2 revealed a heterozygous deletion, c.811-39_841del69bp, at the intron 8/exon 9 junction, in all affected members that was absent from all nonaffected members. RT-PCR analysis and sequencing revealed a novel NF2 transcript characterized by a skipping of exon 9 (75 bp). This deletion is predicted to result in a 25-amino acid deletion in the N-terminal FERM domain of neurofibromin 2. Modeling of this mutant domain suggests possible disorganization of the subdomain C. Conclusion We report the first family with an NF2 mutation associated with intramedullary ependymomas without other features of NF2 syndrome. This mutation, which has not been described previously, may particularly affect the function of neurofibromin 2 in ependymocytes leading to the development of intramedullary WHO grade II ependymomas. We propose that sporadic intramedullary ependymomas should also be analyzed for this region of NF2 gene. PMID:24357459

  9. Analysis of MEFV exon methylation and expression patterns in familial Mediterranean fever

    PubMed Central

    2011-01-01

    Background MEFV mutations and decreased expression level of the gene are related to FMF pathology. DNA methylation at CpG islands is a well-known mechanism for transcriptional silencing. MEFV has a CpG island, spanning a part of the first intron and the whole of the second exon of the gene covering 998 bp region. Here, we tested the hypothesis that the MEFV transcript level in FMF patients correlates with its methylation level, and methylation, by allowing transcription silencing, has a role in FMF ethiopathogenesis. Methods The study group was composed of pediatric FMF patients (N = 51) and age-gender matched healthy controls (N = 21). The relative expression level of MEFV was assessed via quantitative real-time PCR (qRT-PCR) and bisulfite sequencing (BS) was performed to analyse the methylation level quantitatively. Results MEFV expression in FMF patients were decreased compared to healthy controls (P = 0.031). Methylation level of exon 2 of MEFV was found to be slightly higher in FMF patients compared to healthy controls (76% versus 74%) (P = 0.049). The expression level of the MEFV was negatively correlated with the methylation level of the CpG island in both FMF and healthy controls groups (cor = -0.29, P = 0.041) but more so in the FMF only group (cor = -0.36, P = 0.035). Conclusions In this study, the relation between reduced MEFV expression level and FMF was confirmed. Observed slight increase in methylation in FMF patients, and correlation of methylation with expression might be indicative of its role in FMF, however a larger dataset is needed to confirm our preliminary findings. PMID:21819621

  10. Complete exon sequencing of all known Usher syndrome genes greatly improves molecular diagnosis

    PubMed Central

    2011-01-01

    Background Usher syndrome (USH) combines sensorineural deafness with blindness. It is inherited in an autosomal recessive mode. Early diagnosis is critical for adapted educational and patient management choices, and for genetic counseling. To date, nine causative genes have been identified for the three clinical subtypes (USH1, USH2 and USH3). Current diagnostic strategies make use of a genotyping microarray that is based on the previously reported mutations. The purpose of this study was to design a more accurate molecular diagnosis tool. Methods We sequenced the 366 coding exons and flanking regions of the nine known USH genes, in 54 USH patients (27 USH1, 21 USH2 and 6 USH3). Results Biallelic mutations were detected in 39 patients (72%) and monoallelic mutations in an additional 10 patients (18.5%). In addition to biallelic mutations in one of the USH genes, presumably pathogenic mutations in another USH gene were detected in seven patients (13%), and another patient carried monoallelic mutations in three different USH genes. Notably, none of the USH3 patients carried detectable mutations in the only known USH3 gene, whereas they all carried mutations in USH2 genes. Most importantly, the currently used microarray would have detected only 30 of the 81 different mutations that we found, of which 39 (48%) were novel. Conclusions Based on these results, complete exon sequencing of the currently known USH genes stands as a definite improvement for molecular diagnosis of this disease, which is of utmost importance in the perspective of gene therapy. PMID:21569298

  11. Transcriptome networks in the mouse retina: An exon level BXD RI database

    PubMed Central

    King, Rebecca; Lu, Lu; Williams, Robert W.

    2015-01-01

    Purpose Differences in gene expression provide diverse retina phenotypes and may also contribute to susceptibility to injury and disease. The present study defines the transcriptome of the retina in the BXD RI strain set, using the Affymetrix Mouse Gene 2.0 ST array to investigate all exons of traditional protein coding genes, non-coding RNAs, and microRNAs. These data are presented in a highly interactive database on the GeneNetwork website. Methods In the Normal Retina Database, the mRNA levels of the transcriptome from retinas was quantified using the Affymetrix Mouse Gene 2.0 ST array. This database consists of data from male and female mice. The data set includes a total of 52 BXD RI strains, the parental strains (C57BL/6J and DBA/2J), and a reciprocal cross. Results In combination with GeneNetwork, the Department of Defense (DoD) Congressionally Directed Medical Research Programs (CDMRP) Normal Retina Database provides a large resource for mapping, graphing, analyzing, and testing complex genetic networks. Protein-coding and non-coding RNAs can be used to map quantitative trait loci (QTLs) that contribute to expression differences among the BXD strains and to establish links between classical ocular phenotypes associated with differences in the genomic sequence. Using this resource, we extracted transcriptome signatures for retinal cells and defined genetic networks associated with the maintenance of the normal retina. Furthermore, we examined differentially expressed exons within a single gene. Conclusions The high level of variation in mRNA levels found among the BXD RI strains makes it possible to identify expression networks that underline differences in retina structure and function. Ultimately, we will use this database to define changes that occur following blast injury to the retina. PMID:26604663

  12. Evidence for widespread exonic small RNAs in the glaucophyte alga Cyanophora paradoxa.

    PubMed

    Gross, Jeferson; Wajid, Sana; Price, Dana C; Zelzion, Ehud; Li, Junyi; Chan, Cheong Xin; Bhattacharya, Debashish

    2013-01-01

    RNAi (RNA interference) relies on the production of small RNAs (sRNAs) from double-stranded RNA and comprises a major pathway in eukaryotes to restrict the propagation of selfish genetic elements. Amplification of the initial RNAi signal by generation of multiple secondary sRNAs from a targeted mRNA is catalyzed by RNA-dependent RNA polymerases (RdRPs). This phenomenon is known as transitivity and is particularly important in plants to limit the spread of viruses. Here we describe, using a genome-wide approach, the distribution of sRNAs in the glaucophyte alga Cyanophora paradoxa. C. paradoxa is a member of the supergroup Plantae (also known as Archaeplastida) that includes red algae, green algae, and plants. The ancient (>1 billion years ago) split of glaucophytes within Plantae suggests that C. paradoxa may be a useful model to learn about the early evolution of RNAi in the supergroup that ultimately gave rise to plants. Using next-generation sequencing and bioinformatic analyses we find that sRNAs in C. paradoxa are preferentially associated with mRNAs, including a large number of transcripts that encode proteins arising from different functional categories. This pattern of exonic sRNAs appears to be a general trend that affects a large fraction of mRNAs in the cell. In several cases we observe that sRNAs have a bias for a specific strand of the mRNA, including many instances of antisense predominance. The genome of C. paradoxa encodes four sequences that are homologous to RdRPs in Arabidopsis thaliana. We discuss the possibility that exonic sRNAs in the glaucophyte may be secondarily derived from mRNAs by the action of RdRPs. If this hypothesis is confirmed, then transitivity may have had an ancient origin in Plantae. PMID:23844054

  13. Human due diligence.

    PubMed

    Harding, David; Rouse, Ted

    2007-04-01

    Most companies do a thorough job of financial due diligence when they acquire other companies. But all too often, deal makers simply ignore or underestimate the significance of people issues in mergers and acquisitions. The consequences are severe. Most obviously, there's a high degree of talent loss after a deal's announcement. To make matters worse, differences in decision-making styles lead to infighting; integration stalls; and productivity declines. The good news is that human due diligence can help companies avoid these problems. Done early enough, it helps acquirers decide whether to embrace or kill a deal and determine the price they are willing to pay. It also lays the groundwork for smooth integration. When acquirers have done their homework, they can uncover capability gaps, points of friction, and differences in decision making. Even more important, they can make the critical "people" decisions-who stays, who goes, who runs the combined business, what to do with the rank and file-at the time the deal is announced or shortly thereafter. Making such decisions within the first 30 days is critical to the success of a deal. Hostile situations clearly make things more difficult, but companies can and must still do a certain amount of human due diligence to reduce the inevitable fallout from the acquisition process and smooth the integration. This article details the steps involved in conducting human due diligence. The approach is structured around answering five basic questions: Who is the cultural acquirer? What kind of organization do you want? Will the two cultures mesh? Who are the people you most want to retain? And how will rank-and-file employees react to the deal? Unless an acquiring company has answered these questions to its satisfaction, the acquisition it is making will be very likely to end badly. PMID:17432159

  14. Control of calcitonin/calcitonin gene-related peptide pre-mRNA processing by constitutive intron and exon elements.

    PubMed Central

    Yeakley, J M; Hedjran, F; Morfin, J P; Merillat, N; Rosenfeld, M G; Emeson, R B

    1993-01-01

    The calcitonin/calcitonin gene-related peptide (CGRP) primary transcript is alternatively spliced in thyroid C cells and neurons, resulting in the tissue-specific production of calcitonin and CGRP mRNAs. Analyses of mutated calcitonin/CGRP transcription units in permanently transfected cell lines have indicated that alternative splicing is regulated by a differential capacity to utilize the calcitonin-specific splice acceptor. The analysis of an extensive series of mutations suggests that tissue-specific regulation of calcitonin mRNA production does not depend on the presence of a single, unique cis-active element but instead appears to be a consequence of suboptimal constitutive splicing signals. While only those mutations that altered constitutive splicing signals affected splice choices, the action of multiple regulatory sequences cannot be formally excluded. Further, we have identified a 13-nucleotide purine-rich element from a constitutive exon that, when placed in exon 4, entirely switches splice site usage in CGRP-producing cells. These data suggest that specific exon recruitment sequences, in combination with other constitutive elements, serve an important function in exon recognition. These results are consistent with the hypothesis that tissue-specific alternative splicing of the calcitonin/CGRP primary transcript is mediated by cell-specific differences in components of the constitutive splicing machinery. Images PMID:8413203

  15. Massive Idiosyncratic Exon Skipping Corrects the Nonsense Mutation in Dystrophic Mouse Muscle and Produces Functional Revertant Fibers by Clonal Expansion

    PubMed Central

    Lu, Q.L.; Morris, G.E.; Wilton, S.D.; Ly, T.; Artem'yeva, O.V.; Strong, P.; Partridge, T.A.

    2000-01-01

    Conventionally, nonsense mutations within a gene preclude synthesis of a full-length functional protein. Obviation of such a blockage is seen in the mdx mouse, where despite a nonsense mutation in exon 23 of the dystrophin gene, occasional so-called revertant muscle fibers are seen to contain near-normal levels of its protein product. Here, we show that reversion of dystrophin expression in mdx mice muscle involves unprecedented massive loss of up to 30 exons. We detected several alternatively processed transcripts that could account for some of the revertant dystrophins and could not detect genomic deletion from the region commonly skipped in revertant dystrophin. This, together with exon skipping in two noncontiguous regions, favors aberrant splicing as the mechanism for the restoration of dystrophin, but is hard to reconcile with the clonal idiosyncrasy of revertant dystrophins. Revertant dystrophins retain functional domains and mediate plasmalemmal assembly of the dystrophin-associated glycoprotein complex. Physiological function of revertant fibers is demonstrated by the clonal growth of revertant clusters with age, suggesting that revertant dystrophin could be used as a guide to the construction of dystrophin expression vectors for individual gene therapy. The dystrophin gene in the mdx mouse provides a favored system for study of exon skipping associated with nonsense mutations. PMID:10704448

  16. The exon A (C77G) mutation is a common cause of abnormal CD45 splicing in humans.

    PubMed

    Tchilian, E Z; Wallace, D L; Imami, N; Liao, H X; Burton, C; Gotch, F; Martinson, J; Haynes, B F; Beverley, P C

    2001-05-15

    The leukocyte common (CD45) Ag is essential for normal T lymphocyte function and alternative splicing at the N terminus of the gene is associated with changes in T cell maturation and differentiation. Recently, a statistically significant association was reported in a large series of human thymus samples between phenotypically abnormal CD45 splicing and the presence of the CC chemokine receptor 5 deletion 32 (CCR5del32) allele, which confers resistance to HIV infection in homozygotes. We show here that abnormal splicing in these thymus samples is associated with the presence of the only established cause of CD45 abnormal splicing, a C77G transversion in exon A. In addition we have examined 227 DNA samples from peripheral blood of healthy donors and find no association between the exon A (C77G) and CCR5del32 mutations. Among 135 PBMC samples, tested by flow cytometric analysis, all those exhibiting abnormal splicing of CD45 also showed the exon A C77G transversion. We conclude that the exon A (C77G) mutation is a common cause of abnormal CD45 splicing and that further disease association studies of this mutation are warranted. PMID:11342634

  17. Ataxin-3 protein modification as a treatment strategy for spinocerebellar ataxia type 3: removal of the CAG containing exon.

    PubMed

    Evers, Melvin M; Tran, Hoang-Dai; Zalachoras, Ioannis; Pepers, Barry A; Meijer, Onno C; den Dunnen, Johan T; van Ommen, Gert-Jan B; Aartsma-Rus, Annemieke; van Roon-Mom, Willeke M C

    2013-10-01

    Spinocerebellar ataxia type 3 is caused by a polyglutamine expansion in the ataxin-3 protein, resulting in gain of toxic function of the mutant protein. The expanded glutamine stretch in the protein is the result of a CAG triplet repeat expansion in the penultimate exon of the ATXN3 gene. Several gene silencing approaches to reduce mutant ataxin-3 toxicity in this disease aim to lower ataxin-3 protein levels, but since this protein is involved in deubiquitination and proteasomal protein degradation, its long-term silencing might not be desirable. Here, we propose a novel protein modification approach to reduce mutant ataxin-3 toxicity by removing the toxic polyglutamine repeat from the ataxin-3 protein through antisense oligonucleotide-mediated exon skipping while maintaining important wild type functions of the protein. In vitro studies showed that exon skipping did not negatively impact the ubiquitin binding capacity of ataxin-3. Our in vivo studies showed no toxic properties of the novel truncated ataxin-3 protein. These results suggest that exon skipping may be a novel therapeutic approach to reduce polyglutamine-induced toxicity in spinocerebellar ataxia type 3. PMID:23659897

  18. Single nucleotide polymorphism mining and nucleotide sequence analysis of Mx1 gene in exonic regions of Japanese quail

    PubMed Central

    Niraj, Diwesh Kumar; Kumar, Pushpendra; Mishra, Chinmoy; Narayan, Raj; Bhattacharya, Tarun Kumar; Shrivastava, Kush; Bhushan, Bharat; Tiwari, Ashok Kumar; Saxena, Vishesh; Sahoo, Nihar Ranjan; Sharma, Deepak

    2015-01-01

    Aim: An attempt has been made to study the Myxovirus resistant (Mx1) gene polymorphism in Japanese quail. Materials and Methods: In the present, investigation four fragments viz. Fragment I of 185 bp (Exon 3 region), Fragment II of 148 bp (Exon 5 region), Fragment III of 161 bp (Exon 7 region), and Fragment IV of 176 bp (Exon 13 region) of Mx1 gene were amplified and screened for polymorphism by polymerase chain reaction-single-strand conformation polymorphism technique in 170 Japanese quail birds. Results: Out of the four fragments, one fragment (Fragment II) was found to be polymorphic. Remaining three fragments (Fragment I, III, and IV) were found to be monomorphic which was confirmed by custom sequencing. Overall nucleotide sequence analysis of Mx1 gene of Japanese quail showed 100% homology with common quail and more than 80% homology with reported sequence of chicken breeds. Conclusion: The Mx1 gene is mostly conserved in Japanese quail. There is an urgent need of comprehensive analysis of other regions of Mx1 gene along with its possible association with the traits of economic importance in Japanese quail. PMID:27047057

  19. A Targeted Oligonucleotide Enhancer of SMN2 Exon 7 Splicing Forms Competing Quadruplex and Protein Complexes in Functional Conditions

    PubMed Central

    Smith, Lindsay D.; Dickinson, Rachel L.; Lucas, Christian M.; Cousins, Alex; Malygin, Alexey A.; Weldon, Carika; Perrett, Andrew J.; Bottrill, Andrew R.; Searle, Mark S.; Burley, Glenn A.; Eperon, Ian C.

    2014-01-01

    Summary The use of oligonucleotides to activate the splicing of selected exons is limited by a poor understanding of the mechanisms affected. A targeted bifunctional oligonucleotide enhancer of splicing (TOES) anneals to SMN2 exon 7 and carries an exonic splicing enhancer (ESE) sequence. We show that it stimulates splicing specifically of intron 6 in the presence of repressing sequences in intron 7. Complementarity to the 5′ end of exon 7 increases U2AF65 binding, but the ESE sequence is required for efficient recruitment of U2 snRNP. The ESE forms at least three coexisting discrete states: a quadruplex, a complex containing only hnRNP F/H, and a complex enriched in the activator SRSF1. Neither hnRNP H nor quadruplex formation contributes to ESE activity. The results suggest that splicing limited by weak signals can be rescued by rapid exchange of TOES oligonucleotides in various complexes and raise the possibility that SR proteins associate transiently with ESEs. PMID:25263560

  20. Sporadic Hirschsprung`s disease due to a novel nonsense mutation in the RET protooncogene

    SciTech Connect

    Carlson, K.M.; Donis-Keller, H.; Langer, J.C.

    1994-09-01

    Hirschsprung`s disease (HSCR, aganglionic megacolon) is characterized by a lack of ganglion cells along variable lengths of the hindgut. This is most likely due to a failure of the progenitor cells (that are destined to become the ganglion cells of the submucosal and myenteric plexuses) to complete their distal migration in the colon. Recently, mutations in the RET protoocogene have been reported in association with HSCR. We report a novel nonsense mutation resulting in a severely truncated protein. Germline DNA from a panel of 6 HSCR patients was analyzed by SSCP for 20 exons of RET. Eight exons were also directly sequenced. We identified a novel mutation within RET exon 2. The mutation (TAC{sub 36}{yields}TAG{sub 36}), which occurs at nucleotide position 108, involves the replacement of tyrosine with a stop codon and results in a truncated 35 amino acid protein. This mutation is the most 5{prime} nonsense mutation reported thus far. Interestingly, the patient has no prior family history of HSCR and was also diagnosed with multiple developmental anomalies including dysplastic kidney. Recent gene targeting studies with mouse models have shown that RET is essential for normal renal development. However, a parallel phenotype has not been seen in other reported HSCR patients with RET mutations. The observations reported here provide evidence that RET plays a role in human renal development. Ongoing studies will determine the extent of RET involvement in sporadic cases of HSCR.

  1. Evidence for genetic heterogeneity in male pseudohermaphroditism due to Leydig cell hypoplasia.

    PubMed

    Zenteno, J C; Canto, P; Kofman-Alfaro, S; Mendez, J P

    1999-10-01

    Leydig cell aplasia or hypoplasia is a rare form of male pseudohermaphroditism resulting from inadequate fetal testicular Leydig cell differentiation. Affected individuals presented a wide phenotypic spectrum, ranging from complete female external genitalia to males with micropenis. Recessive mutations in the LH receptor gene have been identified as responsible for the condition. The majority of these mutations are point mutations and have been located in exon 11 of the gene. In this study, we report the molecular characterization of the LH receptor gene in three siblings with Leydig cell hypoplasia. After sequencing the 11 exons of the gene, no deleterious mutations were detected in any patient. However, we identified a previously described polymorphism in exon 11. In patients 1 and 3 DNA sequencing revealed a C to T substitution at nucleotide 1065; both patients were homozygous GAT/GAT at codon 355. In contrast, patient 2 was homozygous GAC/GAC, whereas the father and one unaffected sister were heterozygous GAC/GAT at this polymorphic site. These results exclude that Leydig cell hypoplasia in this family is due to a mutation in the LH receptor gene and provide evidence that defects in other loci may also result in failure of Leydig cell differentiation, demonstrating, for the first time, that Leydig cell hypoplasia is a genetically heterogeneous condition. PMID:10523033

  2. Rapid molecular diagnosis of the Gilbert's syndrome-associated exon 1 mutation within the UGT1A1 gene.

    PubMed

    Hsieh, T-Y; Shiu, T-Y; Chu, N-F; Chao, T-Y; Chu, H-C; Chang, W-K; Chao, Y-C; Huang, H-H

    2014-01-01

    Gilbert's syndrome is suspected in patients with unconjugated hyperbilirubinemia caused by decreased activity of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene in the absence of abnormal liver function and hemolysis. The major genetic variants underlying Gilbert's syndrome are TATA-box repeats of the promoter region and exon 1 G211A of the coding region, particularly in Asians. The efficacy of DNA melting curve analysis, however, has not been established for the G211A mutation. For rapid and accurate molecular diagnosis of Gilbert's syndrome, DNA melting curve analysis was evaluated for its genotyping capability not only for TATA-box repeats of the UGT1A1 promoter, but also for G211A of UGT1A1 exon 1. TA repeats within the TATA-box sequence and the exon 1 G211A mutation of the UGT1A1 gene were analyzed by DNA melting curve analysis. To evaluate the assay reliability, direct sequencing or polyacrylamide gel electrophoresis was used as a comparative method. All homozygous and heterozygous polymorphisms of A(TA)7TAA within the TATA-box allele and of exon 1 G211A mutants of the UGT1A1 gene were successfully identified with DNA melting curve analysis. DNA melting curve analysis is, therefore, an effective molecular method for the rapid diagnosis of Gilbert's syndrome, as it detects not only TATA-box polymorphisms but also the exon 1 G211A mutation located within the UGT1A1 gene. PMID:24615032

  3. Isoform switching and exon skipping induced by the DNA methylation inhibitor 5-Aza-2′-deoxycytidine

    PubMed Central

    Ding, Xiao-Lei; Yang, Xiaojing; Liang, Gangning; Wang, Kai

    2016-01-01

    DNA methylation in gene promoters leads to gene silencing and is the therapeutic target of methylation inhibitors such as 5-Aza-2′-deoxycytidine (5-Aza-CdR). By analyzing the time series RNA-seq data (days 5, 9, 13, 17) obtained from human bladder cells exposed to 5-Aza-CdR with 0.1 uM concentration, we showed that 5-Aza-CdR can affect isoform switching and differential exon usage (i.e., exon-skipping), in addition to its effects on gene expression. We identified more than 2,000 genes with significant expression changes after 5-Aza-CdR treatment. Interestingly, 29 exon-skipping events induced by treatment were identified and validated experimentally. Particularly, exon-skipping event in Enhancer of Zeste Homologue 2 (EZH2) along with expression changes showed significant down regulation on Day 5 and Day 9 but returned to normal level on Day 13 and Day 17. EZH2 is a component of the multi-subunit polycomb repressive complex PRC2, and the down-regulation of exon-skipping event may lead to the regain of functional EZH2 which was consistent with our previous finding that demethylation may cause regain of PRC2 in demethylated regions. In summary, our study identified pervasive transcriptome changes of bladder cancer cells after treatment with 5-Aza-CdR, and provided valuable insights into the therapeutic effects of 5-Aza-CdR in current clinical trials. PMID:27090213

  4. Improvement of SMN2 Pre-mRNA Processing Mediated by Exon-Specific U1 Small Nuclear RNA

    PubMed Central

    Dal Mas, Andrea; Rogalska, Malgorzata Ewa; Bussani, Erica; Pagani, Franco

    2015-01-01

    Exon-specific U1 snRNAs (ExSpe U1s) are modified U1 snRNAs that interact with intronic sequences downstream of the 5′ splice site (ss) by complementarity. This process restores exon skipping caused by different types of mutation. We have investigated the molecular mechanism and activity of these molecules in spinal muscular atrophy (SMA), a genetic neuromuscular disease where a silent exonic transition on the survival motor neuron 2 (SMN2) leads to exon 7 (E7) skipping. By using different cellular models, we show that a single chromosome-integrated copy of ExSpe U1 induced a significant correction of endogenous SMN2 E7 splicing and resulted in the restoration of the corresponding SMN protein levels. Interestingly, the analysis of pre-mRNA transcript abundance and decay showed that ExSpe U1s promote E7 inclusion and stabilizes the SMN pre-mRNA intermediate. This selective effect on pre-mRNA stability resulted in higher levels of SMN mRNAs in comparison with those after treatment with an antisense oligonucleotide (AON) that targets corresponding intronic sequences. In mice harboring the SMN2 transgene, AAV-mediated delivery of ExSpe U1 increased E7 inclusion in brain, heart, liver, kidney, and skeletal muscle. The positive effect of ExSpe U1s on SMN pre-mRNA processing highlights their therapeutic potential in SMA and in other pathologies caused by exon-skipping mutations. PMID:25557785

  5. First Report of a Deletion Encompassing an Entire Exon in the Homogentisate 1,2-Dioxygenase Gene Causing Alkaptonuria

    PubMed Central

    Habbal, Mohammad Zouheir; Bou-Assi, Tarek; Zhu, Jun; Owen, Renius; Chehab, Farid F.

    2014-01-01

    Alkaptonuria is often diagnosed clinically with episodes of dark urine, biochemically by the accumulation of peripheral homogentisic acid and molecularly by the presence of mutations in the homogentisate 1,2-dioxygenase gene (HGD). Alkaptonuria is invariably associated with HGD mutations, which consist of single nucleotide variants and small insertions/deletions. Surprisingly, the presence of deletions beyond a few nucleotides among over 150 reported deleterious mutations has not been described, raising the suspicion that this gene might be protected against the detrimental mechanisms of gene rearrangements. The quest for an HGD mutation in a proband with AKU revealed with a SNP array five large regions of homozygosity (5–16 Mb), one of which includes the HGD gene. A homozygous deletion of 649 bp deletion that encompasses the 72 nucleotides of exon 2 and surrounding DNA sequences in flanking introns of the HGD gene was unveiled in a proband with AKU. The nature of this deletion suggests that this in-frame deletion could generate a protein without exon 2. Thus, we modeled the tertiary structure of the mutant protein structure to determine the effect of exon 2 deletion. While the two β-pleated sheets encoded by exon 2 were missing in the mutant structure, other β-pleated sheets are largely unaffected by the deletion. However, nine novel α-helical coils substituted the eight coils present in the native HGD crystal structure. Thus, this deletion results in a deleterious enzyme, which is consistent with the proband’s phenotype. Screening for mutations in the HGD gene, particularly in the Middle East, ought to include this exon 2 deletion in order to determine its frequency and uncover its origin. PMID:25233259

  6. Human GC-AG alternative intron isoforms with weak donor sites show enhanced consensus at acceptor exon positions

    PubMed Central

    Thanaraj, T. A.; Clark, Francis

    2001-01-01

    It has been previously observed that the intrinsically weak variant GC donor sites, in order to be recognized by the U2-type spliceosome, possess strong consensus sequences maximized for base pair formation with U1 and U5/U6 snRNAs. However, variability in signal strength is a fundamental mechanism for splice site selection in alternative splicing. Here we report human alternative GC-AG introns (for the first time from any species), and show that while constitutive GC-AG introns do possess strong signals at their donor sites, a large subset of alternative GC-AG introns possess weak consensus sequences at their donor sites. Surprisingly, this subset of alternative isoforms shows strong consensus at acceptor exon positions 1 and 2. The improved consensus at the acceptor exon can facilitate a strong interaction with U5 snRNA, which tethers the two exons for ligation during the second step of splicing. Further, these isoforms nearly always possess alternative acceptor sites and exhibit particularly weak polypyrimidine tracts characteristic of AG-dependent introns. The acceptor exon nucleotides are part of the consensus required for the U2AF35-mediated recognition of AG in such introns. Such improved consensus at acceptor exons is not found in either normal or alternative GT-AG introns having weak donor sites or weak polypyrimidine tracts. The changes probably reflect mechanisms that allow GC-AG alternative intron isoforms to cope with two conflicting requirements, namely an apparent need for differential splice strength to direct the choice of alternative sites and a need for improved donor signals to compensate for the central mismatch base pair (C-A) in the RNA duplex of U1 snRNA and the pre-mRNA. The other important findings include (i) one in every twenty alternative introns is a GC-AG intron, and (ii) three of every five observed GC-AG introns are alternative isoforms. PMID:11410667

  7. Deletion of Brca2 exon 27 causes hypersensitivity to DNA crosslinks, chromosomal instability, and reduced life span in mice

    NASA Technical Reports Server (NTRS)

    Donoho, Greg; Brenneman, Mark A.; Cui, Tracy X.; Donoviel, Dorit; Vogel, Hannes; Goodwin, Edwin H.; Chen, David J.; Hasty, Paul

    2003-01-01

    The Brca2 tumor-suppressor gene contributes to genomic stability, at least in part by a role in homologous recombinational repair. BRCA2 protein is presumed to function in homologous recombination through interactions with RAD51. Both exons 11 and 27 of Brca2 code for domains that interact with RAD51; exon 11 encodes eight BRC motifs, whereas exon 27 encodes a single, distinct interaction domain. Deletion of all RAD51-interacting domains causes embryonic lethality in mice. A less severe phenotype is seen with BRAC2 truncations that preserve some, but not all, of the BRC motifs. These mice can survive beyond weaning, but are runted and infertile, and die very young from cancer. Cells from such mice show hypersensitivity to some genotoxic agents and chromosomal instability. Here, we have analyzed mice and cells with a deletion of only the RAD51-interacting region encoded by exon 27. Mice homozygous for this mutation (called brca2(lex1)) have a shorter life span than that of control littermates, possibly because of early onsets of cancer and sepsis. No other phenotype was observed in these animals; therefore, the brca2(lex1) mutation is less severe than truncations that delete some BRC motifs. However, at the cellular level, the brca2(lex1) mutation causes reduced viability, hypersensitivity to the DNA interstrand crosslinking agent mitomycin C, and gross chromosomal instability, much like more severe truncations. Thus, the extreme carboxy-terminal region encoded by exon 27 is important for BRCA2 function, probably because it is required for a fully functional interaction between BRCA2 and RAD51. Copyright 2003 Wiley-Liss, Inc.

  8. Correction of Dystrophin Expression in Cells From Duchenne Muscular Dystrophy Patients Through Genomic Excision of Exon 51 by Zinc Finger Nucleases

    PubMed Central

    Ousterout, David G; Kabadi, Ami M; Thakore, Pratiksha I; Perez-Pinera, Pablo; Brown, Matthew T; Majoros, William H; Reddy, Timothy E; Gersbach, Charles A

    2015-01-01

    Duchenne muscular dystrophy (DMD) is caused by genetic mutations that result in the absence of dystrophin protein expression. Oligonucleotide-induced exon skipping can restore the dystrophin reading frame and protein production. However, this requires continuous drug administration and may not generate complete skipping of the targeted exon. In this study, we apply genome editing with zinc finger nucleases (ZFNs) to permanently remove essential splicing sequences in exon 51 of the dystrophin gene and thereby exclude exon 51 from the resulting dystrophin transcript. This approach can restore the dystrophin reading frame in ~13% of DMD patient mutations. Transfection of two ZFNs targeted to sites flanking the exon 51 splice acceptor into DMD patient myoblasts led to deletion of this genomic sequence. A clonal population was isolated with this deletion and following differentiation we confirmed loss of exon 51 from the dystrophin mRNA transcript and restoration of dystrophin protein expression. Furthermore, transplantation of corrected cells into immunodeficient mice resulted in human dystrophin expression localized to the sarcolemmal membrane. Finally, we quantified ZFN toxicity in human cells and mutagenesis at predicted off-target sites. This study demonstrates a powerful method to restore the dystrophin reading frame and protein expression by permanently deleting exons. PMID:25492562

  9. Union Exon Based Approach for RNA-Seq Gene Quantification: To Be or Not to Be?

    PubMed Central

    Zhao, Shanrong; Xi, Li; Zhang, Baohong

    2015-01-01

    In recent years, RNA-seq is emerging as a powerful technology in estimation of gene and/or transcript expression, and RPKM (Reads Per Kilobase per Million reads) is widely used to represent the relative abundance of mRNAs for a gene. In general, the methods for gene quantification can be largely divided into two categories: transcript-based approach and ‘union exon’-based approach. Transcript-based approach is intrinsically more difficult because different isoforms of the gene typically have a high proportion of genomic overlap. On the other hand, ‘union exon’-based approach method is much simpler and thus widely used in RNA-seq gene quantification. Biologically, a gene is expressed in one or more transcript isoforms. Therefore, transcript-based approach is logistically more meaningful than ‘union exon’-based approach. Despite the fact that gene quantification is a fundamental task in most RNA-seq studies, however, it remains unclear whether ‘union exon’-based approach for RNA-seq gene quantification is a good practice or not. In this paper, we carried out a side-by-side comparison of ‘union exon’-based approach and transcript-based method in RNA-seq gene quantification. It was found that the gene expression levels are significantly underestimated by ‘union exon’-based approach, and the average of RPKM from ‘union exons’-based method is less than 50% of the mean expression obtained from transcript-based approach. The difference between the two approaches is primarily affected by the number of transcripts in a gene. We performed differential analysis at both gene and transcript levels, respectively, and found more insights, such as isoform switches, are gained from isoform differential analysis. The accuracy of isoform quantification would improve if the read coverage pattern and exon-exon spanning reads are taken into account and incorporated into EM (Expectation Maximization) algorithm. Our investigation discourages the use of

  10. Mechanistic Evaluation for Mixed-field Agglutination in the K562 Cell Study Model with Exon 3 Deletion of A1 Gene.

    PubMed

    Chen, Ding-Ping; Tseng, Ching-Ping; Lin, Chi-Jui; Wang, Wei-Ting; Sun, Chien-Feng

    2015-01-01

    In the case of blood type B3 with typical mixed-field agglutination of RBCs in the presence of anti-B or anti-AB antibody, a number of genetic alternations have been reported. It is well known that the IVS3+5G→A mutation in the B gene destroys the consensus of the splice donor site leading to exon 3 skipping during mRNA splicing. The lack of exon 3 likely causes a short stem region, producing an unstable B3 protein, and is concomitant with a decrease in B3 protein expression. Whether the phenomenon also appears in the type A blood group is of question. In this study, we evaluate whether exon 3 deletion in the blood type A gene also results in mixed-field phenotype. Site-directed mutagenesis was used to generate cDNA encoding A1 gene with exon 3 deletion. The cDNA was stably expressed in K562 cells. The expression of A antigen was compared with expression in parental K562 cells that did not express A antigen and in the stable K562 cell line expressing A(1) cDNA by flow cytometry analyses. The expression of A antigen in A1 stable cells and parental K562 cells was set as 100% and 0%, respectively. The mean relative percentage of A antigen expression for the cells of A1 with exon 3 deletion was 59.9% of A1 stable cells. Consistent with the observations of B3, which is B gene with exon 3 deletion, mixed field agglutination was observed for the cells expressing A1 with exon 3 deletion. Exon 3 deletion results in mixed field phenotype in both type A and B RBCs. However, the degree of antigen expression change for exon 3 deletion in A gene was less severe when compared with the deletion occurred in B gene. PMID:26663798

  11. Epidermal growth factor receptor‐tyrosine kinase inhibitor therapy is especially beneficial to patients with exon 19 deletion compared with exon 21 L858R mutation in non‐small‐cell lung cancer: Systematic review and meta analysis

    PubMed Central

    Liu, Yinghui; Ren, Zuen; Wang, Jinghui

    2016-01-01

    Abstract Background The correlation between epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) and EGFR sensitive mutation subtypes in advanced or metastatic non‐small cell lung cancer (NSCLC) remains uncertain. We performed this meta‐analysis to determine different clinical outcomes between patients with exon 19 deletion accepting EGFR‐TKI therapy compared with those with exon 21 L858R mutation. Methods PubMed and Web of Science were analyzed for eligible trials. Raw data were extracted to give pooled estimates of the effect of EGFR‐TKI therapy on objective response rate (ORR), one‐year progression‐free survival (PFS), and two‐year overall survival (OS). Results We identified 13 eligible trials involving 912 patients. Prospective meta‐analysis demonstrated that the ORR of the 19 deletion group was significantly higher than the 21 L858R mutation group (odds ratio [OR] 1.98, 95% confidence interval [CI] 1.18–3.33; P = 0.01), but no statistical significance between the one‐year PFS rate of the 19 deletion and 21 L858R groups (OR 1.44, 95% CI 0.96–2.18; P = 0.08) was found. However, retrospective meta‐analysis demonstrated that a significantly higher one‐year PFS rate was associated with the 19 deletion group (OR 1.73, 95% CI 1.17–2.56; P = 0.006). The two‐year survival rate of the 19 deletion group was significantly higher than the 21 L858R group (OR 5.27, 95 % CI 1.76–15.71; P = 0.003). Conclusions In advanced NSCLC patients, an exon 19 deleton may provide superior ORR, PFS, and OS after EGFR‐TKI treatment compared with an exon 21 L858R mutation. PMID:27385982

  12. Clinical features of patients with dystrophinopathy sharing the 45-55 exon deletion of DMD gene.

    PubMed

    Taglia, Antonella; Petillo, Roberta; D'Ambrosio, Paola; Picillo, Esther; Torella, Annalaura; Orsini, Chiara; Ergoli, Manuela; Scutifero, Marianna; Passamano, Luigia; Palladino, Alberto; Nigro, Gerardo; Politano, Luisa

    2015-05-01

    Becker muscular dystrophy (BMD) was first described in 1953 by Emile Becker as a benign variant of Duchenne muscular Dystrophy (DMD). Compared with DMD, BMD is clinically more heterogeneous, with initial presentation in the teenage years and loss of ambulation beyond the age of 16 and a wide spectrum of clinical presentations, ranging from only myalgias and muscle cramps to exercise intolerance and myoglobinuria, asymptomatic elevation of serum creatin-kinase, or mild limb-girdle weakness and quadriceps myopathy. About 50% of patients become symptomatic by the age of 10 and the most part by the age of 20 years. However few patients can be free of symptoms till their fifties and cases of late-onset Becker Muscular Dystrophy have also been described. In this report we describe the clinical features of patients with dystrophinopathy sharing a deletion of exons 45-55, occasionally or retrospectively diagnosed. These data are important for both the prognostic aspects of children presenting this dystrophin gene mutation, and for the genetic counseling in these families (reassuring them on the benign course of the disease), and last but not least to keep in mind a diagnosis of BMD in asymptomatic adults with mild hyperckemia. PMID:26155064

  13. Haploinsufficiency for Core Exon Junction Complex Components Disrupts Embryonic Neurogenesis and Causes p53-Mediated Microcephaly.

    PubMed

    Mao, Hanqian; McMahon, John J; Tsai, Yi-Hsuan; Wang, Zefeng; Silver, Debra L

    2016-09-01

    The exon junction complex (EJC) is an RNA binding complex comprised of the core components Magoh, Rbm8a, and Eif4a3. Human mutations in EJC components cause neurodevelopmental pathologies. Further, mice heterozygous for either Magoh or Rbm8a exhibit aberrant neurogenesis and microcephaly. Yet despite the requirement of these genes for neurodevelopment, the pathogenic mechanisms linking EJC dysfunction to microcephaly remain poorly understood. Here we employ mouse genetics, transcriptomic and proteomic analyses to demonstrate that haploinsufficiency for each of the 3 core EJC components causes microcephaly via converging regulation of p53 signaling. Using a new conditional allele, we first show that Eif4a3 haploinsufficiency phenocopies aberrant neurogenesis and microcephaly of Magoh and Rbm8a mutant mice. Transcriptomic and proteomic analyses of embryonic brains at the onset of neurogenesis identifies common pathways altered in each of the 3 EJC mutants, including ribosome, proteasome, and p53 signaling components. We further demonstrate all 3 mutants exhibit defective splicing of RNA regulatory proteins, implying an EJC dependent RNA regulatory network that fine-tunes gene expression. Finally, we show that genetic ablation of one downstream pathway, p53, significantly rescues microcephaly of all 3 EJC mutants. This implicates p53 activation as a major node of neurodevelopmental pathogenesis following EJC impairment. Altogether our study reveals new mechanisms to help explain how EJC mutations influence neurogenesis and underlie neurodevelopmental disease. PMID:27618312

  14. A genome-wide analysis of putative functional and exonic variation associated with extremely high intelligence

    PubMed Central

    Kadeva, Neli; Miller, Mike B.; Iacono, William G.; McGue, Matt; Stergiakouli, Evie; Davey Smith, George; Putallaz, Martha; Lubinski, David; Meaburn, Emma L.; Plomin, Robert; Simpson, Michael A.

    2015-01-01

    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. The present 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 1 409 individuals drawn from the top 0.0003 (IQ > 170) of the population distribution of intelligence and 3 253 unselected population-based controls. Our analysis focused on putative functional exonic variants assayed on the Illumina Human Exome 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% (SE 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. PMID:26239293

  15. A Single Divergent Exon Inhibits Ankyrin-B Association with the Plasma Membrane

    PubMed Central

    He, Meng; Tseng, Wei-Chou; Bennett, Vann

    2013-01-01

    Vertebrate ankyrin-B and ankyrin-G exhibit divergent subcellular localization and function despite their high sequence and structural similarity and common origin from a single ancestral gene at the onset of chordate evolution. Previous studies of ankyrin family diversity have focused on the C-terminal regulatory domain. Here, we identify an ankyrin-B-specific linker peptide connecting the ankyrin repeat domain to the ZU52-UPA module that inhibits binding of ankyrin-B to membrane protein partners E-cadherin and neurofascin 186 and prevents association of ankyrin-B with epithelial lateral membranes as well as neuronal plasma membranes. The residues of the ankyrin-B linker required for autoinhibition are encoded by a small exon that is highly divergent between ankyrin family members but conserved in the ankyrin-B lineage. We show that the ankyrin-B linker suppresses activity of the ANK repeat domain through an intramolecular interaction, likely with a groove on the surface of the ANK repeat solenoid, thereby regulating the affinities between ankyrin-B and its binding partners. These results provide a simple evolutionary explanation for how ankyrin-B and ankyrin-G have acquired striking differences in their plasma membrane association while maintaining overall high levels of sequence similarity. PMID:23569209

  16. Folding Factors and Partners for the Intrinsically Disordered Protein Micro-Exon Gene 14 (MEG-14)

    PubMed Central

    Lopes, Jose Luiz S.; Orcia, Debora; Araujo, Ana Paula U.; DeMarco, Ricardo; Wallace, B.A.

    2013-01-01

    The micro-exon genes (MEG) of Schistosoma mansoni, a parasite responsible for the second most widely spread tropical disease, code for small secreted proteins with sequences unique to the Schistosoma genera. Bioinformatics analyses suggest the soluble domain of the MEG-14 protein will be largely disordered, and using synchrotron radiation circular dichroism spectroscopy, its secondary structure was shown to be essentially completely unfolded in aqueous solution. It does, however, show a strong propensity to fold into more ordered structures under a wide range of conditions. Partial folding was produced by increasing temperature (in a reversible process), contrary to the behavior of most soluble proteins. Furthermore, significant folding was observed in the presence of negatively charged lipids and detergents, but not in zwitterionic or neutral lipids or detergents. Absorption onto a surface followed by dehydration stimulated it to fold into a helical structure, as it did when the aqueous solution was replaced by nonaqueous solvents. Hydration of the dehydrated folded protein was accompanied by complete unfolding. These results support the identification of MEG-14 as a classic intrinsically disordered protein, and open the possibility of its interaction/folding with different partners and factors being related to multifunctional roles and states within the host. PMID:23746524

  17. Frequency of HLA-G exon 8 polymorphisms and kidney allograft outcome in Iranian population.

    PubMed

    Aghdaie, Mahdokht H; Azarpira, Negar; Kazemi, Kurosh; Geramizadeh, Bita; Darai, Masumeh; Malekhoseini, Seid Ali

    2011-06-01

    The 14-bp polymorphism in exon 8 of the HLA-G gene is associated with HLA-G mRNA stability and the patterns of alternative isoform splicing and may influence the functionality of the HLA-G molecule. HLA-G expression was related to allograft acceptance and fewer episodes of acute rejection during heart, kidney and liver-kidney transplantation. In order to determine a possible correlation between the 14-bp insertion/deletion polymorphism and kidney allograft outcome in our population, genomic DNA was isolated from 144 patients who had received isolated kidney allografts. The recipients was divided into two groups, grafts presenting features of rejection group and a non-rejection group, and compared them with a control group of 100 healthy subjects. There was no significant difference in allelic frequencies of 14-bp insertion/deletion polymorphism between normal controls and kidney transplant patients. No significant difference was found between the RG and the NRG regarding the 14-bp genotypes and alleles. Therefore, additional studies with more sample size from other populations with analysis of other HLA-G polymorphisms are necessary to define this polymorphism as a valuable clinical marker. PMID:21107725

  18. ADHD and the DRD4 exon III 7-repeat polymorphism: an international meta-analysis

    PubMed Central

    Gray, Jeremy R.

    2010-01-01

    We sought to elucidate the relationship of ADHD (Attention-Deficit Hyperactivity Disorder) to the DRD4 exon III VNTR 7R allele worldwide using analytic techniques and to relate these findings to the field of cultural neuroscience. To focus on a potential moderating role of race/ethnicity, we excluded over 30 papers that have explored the relationship between the DRD4 7R and ADHD but had unclear or lax racial–ethnic inclusion criteria. The papers in this meta-analysis were only included if a single race made up 95% or more of their sample. We searched for and translated papers not published in English, and found a significant difference in the relationship of ADHD and DRD4 7R in people of European-Caucasian (Odds ratio 1.635, Z = 3.936, P < 0.00001) and South American (Odds ratio 2.407, Z = 3.317, P = 0.001) descent vs people of Middle Eastern ancestry (Odds ratio 0.717, Z = −2.466; P = 0.014). We also examined the moderating effect of differing ADHD diagnoses, subject recruitment, control recruitment and male to female ratio. Finally, we consider the implications of these data for cultural neuroscience. PMID:20019071

  19. Alternative Splicing and Transcriptome Profiling of Experimental Autoimmune Encephalomyelitis Using Genome-Wide Exon Arrays

    PubMed Central

    Gillett, Alan; Maratou, Klio; Fewings, Chris; Harris, Robert A.; Jagodic, Maja; Aitman, Tim; Olsson, Tomas

    2009-01-01

    Background Multiple Sclerosis (MS) is a chronic inflammatory disease causing demyelination and nerve loss in the central nervous system. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS that is widely used to investigate complex pathogenic mechanisms. Transcriptional control through isoform selection and mRNA levels determines pathway activation and ultimately susceptibility to disease. Methodology/Principal Findings We have studied the role of alternative splicing and differential expression in lymph node cells from EAE-susceptible Dark Agouti (DA) and EAE-resistant Piebald Virol Glaxo.AV1 (PVG) inbred rat strains using Affymetrix Gene Chip Rat Exon 1.0 ST Arrays. Comparing the two strains, we identified 11 differentially spliced and 206 differentially expressed genes at day 7 post-immunization, as well as 9 differentially spliced and 144 differentially expressed genes upon autoantigen re-stimulation. Functional clustering and pathway analysis implicate genes for glycosylation, lymphocyte activation, potassium channel activity and cellular differentiation in EAE susceptibility. Conclusions/Significance Our results demonstrate that alternative splicing occurs during complex disease and may govern EAE susceptibility. Additionally, transcriptome analysis not only identified previously defined EAE pathways regulating the immune system, but also novel mechanisms. Furthermore, several identified genes overlap known quantitative trait loci, providing novel causative candidate targets governing EAE. PMID:19915720

  20. Polyglutamine disruption of the huntingtin exon1 N-terminus triggers a complex aggregation mechanism

    PubMed Central

    Thakur, Ashwani K.; Jayaraman, Murali; Mishra, Rakesh; Thakur, Monika; Chellgren, Veronique M.; Byeon, In-Ja; Anjum, Dalaver H.; Kodali, Ravindra; Creamer, Trevor P.; Conway, James F.; M.Gronenborn, Angela; Wetzel, Ronald

    2009-01-01

    Simple polyglutamine (polyQ) peptides aggregate in vitro via a nucleated growth pathway directly yielding amyloid-like aggregates. We show here that the 17 amino acid flanking sequence (httNT) N-terminal to the polyQ in the toxic huntingtin exon1 fragment imparts onto this peptide a complex alternative aggregation mechanism. In isolation the httNT peptide is a compact coil that resists aggregation. When polyQ is fused to this sequence, it induces in httNT, in a repeat-length dependent fashion, a more extended conformation that greatly enhances its aggregation into globular oligomers with httNT cores and exposed polyQ. In a second step, a new, amyloid-like aggregate is formed with a core composed of both httNT and polyQ. The results indicate unprecedented complexity in how primary sequence controls aggregation within a substantially disordered peptide, and have implications for the molecular mechanism of Huntington's disease. PMID:19270701

  1. An exon splice enhancer primes IGF2:IGF2R binding site structure and function evolution.

    PubMed

    Williams, Christopher; Hoppe, Hans-Jürgen; Rezgui, Dellel; Strickland, Madeleine; Forbes, Briony E; Grutzner, Frank; Frago, Susana; Ellis, Rosamund Z; Wattana-Amorn, Pakorn; Prince, Stuart N; Zaccheo, Oliver J; Nolan, Catherine M; Mungall, Andrew J; Jones, E Yvonne; Crump, Matthew P; Hassan, A Bassim

    2012-11-30

    Placental development and genomic imprinting coevolved with parental conflict over resource distribution to mammalian offspring. The imprinted genes IGF2 and IGF2R code for the growth promoter insulin-like growth factor 2 (IGF2) and its inhibitor, mannose 6-phosphate (M6P)/IGF2 receptor (IGF2R), respectively. M6P/IGF2R of birds and fish do not recognize IGF2. In monotremes, which lack imprinting, IGF2 specifically bound M6P/IGF2R via a hydrophobic CD loop. We show that the DNA coding the CD loop in monotremes functions as an exon splice enhancer (ESE) and that structural evolution of binding site loops (AB, HI, FG) improved therian IGF2 affinity. We propose that ESE evolution led to the fortuitous acquisition of IGF2 binding by M6P/IGF2R that drew IGF2R into parental conflict; subsequent imprinting may then have accelerated affinity maturation. PMID:23197533

  2. Characterization of 5' promoter and exon 1-3 polymorphism of the RAET1E gene.

    PubMed

    Cox, Steven T; Pearson, Hayley; Laza-Briviesca, Raquel; Pesoa, Susanna; Vullo, Carlos; Madrigal, J Alejandro; Saudemont, Aurore

    2016-01-01

    NKG2D is an activating receptor utilized by natural killer (NK) cells that recognizes upregulated ligands on infected, tumorigenic and damaged cells, leading to their cytolysis. However, the NKG2D ligand (NKG2DL) system is very complex with eight known gene loci encoding slightly different molecules. Furthermore, most NKG2DL gene loci such as MICA and MICB are highly polymorphic with potential for functional differences. NKG2DL expression on tumors varies depending on the malignancy and tumors can also release soluble NKG2DL that exert anergic effects on NK cells when engagement with NKG2D occurs, allowing escape from NK cell immunosurveillance. We carried out RAET1E typing of IHW cell line DNA, including a 580 bp proximal promoter fragment and exons 1-3 identifying 13 of 15 known RAET1E alleles. We determined 7 polymorphisms within the promoter region, including 2 already known that contributed to 9 promoter types. RAET1E alleles with variability in the extracellular region also differed with respect to promoter type and one allele, RAET1E(∗)003, associated with 5 promoter types. We then identified putative transcription factor binding sites for RAET1E, and found 5 of the 7 promoter polymorphisms may disrupt these sites, abrogating binding of transcription factors and varying the potential level of expression. PMID:26519211

  3. A genome-wide analysis of putative functional and exonic variation associated with extremely high intelligence.

    PubMed

    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. PMID:26239293

  4. Perispeckles are major assembly sites for the exon junction core complex

    PubMed Central

    Daguenet, Elisabeth; Baguet, Aurélie; Degot, Sébastien; Schmidt, Ute; Alpy, Fabien; Wendling, Corinne; Spiegelhalter, Coralie; Kessler, Pascal; Rio, Marie-Christine; Le Hir, Hervé; Bertrand, Edouard; Tomasetto, Catherine

    2012-01-01

    The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites. PMID:22419818

  5. Perispeckles are major assembly sites for the exon junction core complex.

    PubMed

    Daguenet, Elisabeth; Baguet, Aurélie; Degot, Sébastien; Schmidt, Ute; Alpy, Fabien; Wendling, Corinne; Spiegelhalter, Coralie; Kessler, Pascal; Rio, Marie-Christine; Le Hir, Hervé; Bertrand, Edouard; Tomasetto, Catherine

    2012-05-01

    The exon junction complex (EJC) is loaded onto mRNAs as a consequence of splicing and regulates multiple posttranscriptional events. MLN51, Magoh, Y14, and eIF4A3 form a highly stable EJC core, but where this tetrameric complex is assembled in the cell remains unclear. Here we show that EJC factors are enriched in domains that we term perispeckles and are visible as doughnuts around nuclear speckles. Fluorescence resonance energy transfer analyses and EJC assembly mutants show that perispeckles do not store free subunits, but instead are enriched for assembled cores. At the ultrastructural level, perispeckles are distinct from interchromatin granule clusters that may function as storage sites for splicing factors and intermingle with perichromatin fibrils, where nascent RNAs and active RNA Pol II are present. These results support a model in which perispeckles are major assembly sites for the tetrameric EJC core. This subnuclear territory thus represents an intermediate region important for mRNA maturation, between transcription sites and splicing factor reservoirs and assembly sites. PMID:22419818

  6. The DRD4 exon 3 VNTR polymorphism and addiction-related phenotypes: a review

    PubMed Central

    McGeary, John

    2009-01-01

    In addition to the large literatures on associations of the DRD4 VNTR polymorphism with ADHD and personality traits, there is an emerging literature linking this variant to addiction and addiction-related phenotypes. When only diagnosis-based studies are considered, an inconsistent picture emerges raising doubts as to the relevance of this polymorphism to addiction. However the use of multiple levels of analysis in examining the importance of this polymorphism has raised the possibility of an urge-related “intermediate phenotype” that puts one at risk for developing addiction but may not be found in all persons with an addiction diagnosis. From cellular assays through neuroimaging and behavioral phenotypes, these studies highlight the power of the “intermediate phenotype” approach and suggest a possible explanation of the mixed findings when diagnosis is used as the phenotype. Strengths and weaknesses of alternative DRD4 VNTR genotype grouping strategies are discussed. In sum, converging evidence across multiple methodologies supports the possibility of a robust relationship between the DRD4 exon 3 VNTR polymorphism and urge for addictive substances. PMID:19336242

  7. Live Cell Genomics: RNA Exon-Specific RNA-Binding Protein Isolation.

    PubMed

    Bell, Thomas J; Eberwine, James

    2015-01-01

    RNA-binding proteins (RBPs) are essential regulatory proteins that control all modes of RNA processing and regulation. New experimental approaches to isolate these indispensable proteins under in vivo conditions are needed to advance the field of RBP biology. Historically, in vitro biochemical approaches to isolate RBP complexes have been useful and productive, but biological relevance of the identified RBP complexes can be imprecise or erroneous. Here we review an inventive experimental to isolate RBPs under the in vivo conditions. The method is called peptide nucleic acid (PNA)-assisted identification of RBP (PAIR) technology and it uses cell-penetrating peptides (CPPs) to deliver photo-activatible RBP-capture molecule to the cytoplasm of the live cells. The PAIR methodology provides two significant advantages over the most commonly used approaches: (1) it overcomes the in vitro limitation of standard biochemical approaches and (2) the PAIR RBP-capture molecule is highly selective and adaptable which allows investigators to isolate exon-specific RBP complexes. Most importantly, the in vivo capture conditions and selectivity of the RBP-capture molecule yield biologically accurate and relevant RBP data. PMID:26202289

  8. One missense mutation in exon 2 of the PAX5 gene in Iran.

    PubMed

    Yazdanparast, S; Khatami, S R; Galehdari, H; Jaseb, K

    2015-01-01

    The PAX5 gene, which encodes the B-cell specific activator protein, is one of the most important factors in determination of B-cell development. This gene is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). For example, point mutations, deletions, as well as other gene rearrangements may lead to several forms of B-cell malignancy. In this study, we obtained 50 blood samples from patients diagnosed with ALL, and screened for PAX5 mutations using sequencing in exons 1, 2 and 3. We found a heterozygous germline variant, c.113G>A (p.Arg38His), which affects the paired domain of PAX5. It seems that this mutation is pathogenic, but is recessive. Our findings suggest that this mutation in a single allele of the PAX5 gene is not sufficient to cause disease, and it is possible that other alleles are also involved in the onset of B-ALL. PMID:26782422

  9. Lack of exonic sulfotransferase 4A1 mutations in controls and schizophrenia cases.

    PubMed

    Lewis, Aaron G; Minchin, Rodney F

    2009-02-01

    Sulfotransferase 4A1 (SULT4A1) is a novel sulfotransferase expressed almost exclusively in the brain. The gene is located on chromosome 22q13.3, a region implicated in predisposition to schizophrenia. Recently, a variable microsatellite region located upstream of SULT4A1 was found to be associated with an increase in schizophrenia risk. We hypothesised that if functional dysregulation of SULT4A1 was involved in the aetiology of schizophrenia, then genetic variants in the coding sequence of SULT4A1 might be identified in cases compared with controls. To test this, we carried out a mutation analysis of the coding region (exons 2-7) in 71 Australian schizophrenia cases and 69 controls. We found no mutations, either synonymous or nonsynonymous, in either cohort. However, intronic variants (IVS5+12 C>T and IVS5+28 G>C) were identified, the frequency of which was not statistically different between cases and controls. The lack of polymorphisms in the coding region of the SULT4A1 gene is highly unusual and, along with its high conservation between species, suggests that SULT4A1 may have an important function in vivo. However, our findings do not support the hypothesis that germline mutations in the coding region of SULT4A1 contribute to susceptibility to schizophrenia. PMID:19125109

  10. Distal Alternative Last Exons Localize mRNAs to Neural Projections.

    PubMed

    Taliaferro, J Matthew; Vidaki, Marina; Oliveira, Ruan; Olson, Sara; Zhan, Lijun; Saxena, Tanvi; Wang, Eric T; Graveley, Brenton R; Gertler, Frank B; Swanson, Maurice S; Burge, Christopher B

    2016-03-17

    Spatial restriction of mRNA to distinct subcellular locations enables local regulation and synthesis of proteins. However, the organizing principles of mRNA localization remain poorly understood. Here we analyzed subcellular transcriptomes of neural projections and soma of primary mouse cortical neurons and two neuronal cell lines and found that alternative last exons (ALEs) often confer isoform-specific localization. Surprisingly, gene-distal ALE isoforms were four times more often localized to neurites than gene-proximal isoforms. Localized isoforms were induced during neuronal differentiation and enriched for motifs associated with muscleblind-like (Mbnl) family RNA-binding proteins. Depletion of Mbnl1 and/or Mbnl2 reduced localization of hundreds of transcripts, implicating Mbnls in localization of mRNAs to neurites. We provide evidence supporting a model in which the linkage between genomic position of ALEs and subcellular localization enables coordinated induction of localization-competent mRNA isoforms through a post-transcriptional regulatory program that is induced during differentiation and reversed in cellular reprogramming and cancer. PMID:26907613

  11. INI1 mutations in meningiomas at a potential hotspot in exon 9

    PubMed Central

    Schmitz, U; Mueller, W; Weber, M; Sévenet, N; Delattre, O; Deimling, A von

    2001-01-01

    Rhabdoid tumours have been shown to carry somatic mutations in the INI1 (SMARCB1/hSNF5) gene. A considerable fraction of these tumours exhibit allelic losses on chromosome 22. Allelic loss on 22q also is characteristic for meningiomas, however most of these alterations are considered to be associated with mutations of the NF2 gene. We examined a series of 126 meningiomas for alterations in the INI1 gene. Four identical somatic mutations in exon 9 were detected resulting in an exchange of Arg to His in position 377 of INI1. Our observations were reproduced both by using DNA from a new round of extraction and by employing overlapping primers. This mutational hotspot therefore appears to be an important target in the formation of a fraction of meningiomas. In addition, 4 novel polymorphisms of INI1 were characterized. Our data indicate that the INI1 is a second tumour suppressor gene on chromosome 22 that may be important for the genesis of meningiomas. © 2001 Cancer Research Campaign http://www.bjcancer.com PMID:11161377

  12. Somatic mutational analysis of MED12 exon 2 in uterine leiomyomas of Iranian women

    PubMed Central

    Shahbazi, Shirin; Fatahi, Neda; Amini-Moghaddam, Soheila

    2015-01-01

    Uterine leiomyomas are steroid-hormone dependent tumors of myometrial smooth muscle cells that affect numerous women throughout the world. Based on previous studies, we evaluated the mutations of MED12 gene which encodes a co-activator protein involved in transcription regulation of the vast majority of RNA polymerase II-dependent genes. Exon 2 of MED12 gene was genotyped by PCR-sequencing method. To determine the proportion of mutation-containing transcripts, RNA was extracted from the tissue samples and the corresponding amplified cDNA was sequenced. We observed 11 mutation positive lesions, 7 of them were located in codon 44. The c.131G>A was found to be the most common somatic mutation in this study. Our investigation also demonstrated two unreported mutations , one large deletion and one insertion. cDNA analyzing revealed that the mutated transcripts were predominantly expressed in almost all changes including the new insertion mutation c.122-123ins15. Our study provides further evidence that the MED12 somatic mutations occur in a heterozygous manner and are mostly missense mutations in codon 44. The results displayed 47.8% mutation positive lesions in Iranian patients confirming the diversity between the populations. PMID:26396919

  13. Prion protein gene (PRNP) variants and evidence for strong purifying selection in functionally important regions of bovine exon 3

    PubMed Central

    Seabury, Christopher M.; Honeycutt, Rodney L.; Rooney, Alejandro P.; Halbert, Natalie D.; Derr, James N.

    2004-01-01

    Amino acid replacements encoded by the prion protein gene (PRNP) have been associated with transmissible and hereditary spongiform encephalopathies in mammalian species. However, an association between bovine spongiform encephalopathy (BSE) and bovine PRNP exon 3 has not been detected. Moreover, little is currently known regarding the mechanisms of evolution influencing the bovine PRNP gene. Therefore, in this study we evaluated the patterns of nucleotide variation associated with PRNP exon 3 for 36 breeds of domestic cattle and representative samples for 10 additional species of Bovinae. The results of our study indicate that strong purifying selection has intensely constrained PRNP over the long-term evolutionary history of the subfamily Bovinae, especially in regions considered to be of functional, structural, and pathogenic importance in humans as well as other mammals. The driving force behind this intense level of purifying selection remains to be explained. PMID:15477588

  14. Familial occurrence of typical and severe lethal congenital contractural arachnodactyly caused by missplicing of exon 34 of fibrillin-2

    SciTech Connect

    Wang, Mei; Godfrey, M.; Clericuzio, C.L.

    1996-11-01

    Genetic linkage studies have linked congenital contractual arachnodactyly (CCA), a usually mild heritable connective-tissue disorder, to FBN2, the fibrillin gene on chromosome 5. Recently, FBN2 mutations in two patients with CCA have been described. Here we report an A{r_arrow}T transversion at the -2 position of the consensus acceptor splice site, resulting in the missplicing of exon 34, a calcium-binding epidermal growth factor-like repeat in fibrillin-2 in a mother and daughter with CCA. Significantly, the mother exhibited a classic CCA phenotype with arachnodactyly, joint contractures, and abnormal pinnae, whereas her daughter exhibited a markedly more severe CCA phenotype, which included cardiovascular and gastrointestinal anomalies that led to death in infancy. Analysis of cloned fibroblasts showed that the mother is a somatic mosaic for the exon 34 missplicing mutation, whereas all the daughter`s cells harbored the mutation. 48 refs., 6 figs., 2 tabs.

  15. Recurrent Loss of NFE2L2 Exon 2 Is a Mechanism for Nrf2 Pathway Activation in Human Cancers.

    PubMed

    Goldstein, Leonard D; Lee, James; Gnad, Florian; Klijn, Christiaan; Schaub, Annalisa; Reeder, Jens; Daemen, Anneleen; Bakalarski, Corey E; Holcomb, Thomas; Shames, David S; Hartmaier, Ryan J; Chmielecki, Juliann; Seshagiri, Somasekar; Gentleman, Robert; Stokoe, David

    2016-09-01

    The Nrf2 pathway is frequently activated in human cancers through mutations in Nrf2 or its negative regulator KEAP1. Using a cell-line-derived gene signature for Nrf2 pathway activation, we found that some tumors show high Nrf2 activity in the absence of known mutations in the pathway. An analysis of splice variants in oncogenes revealed that such tumors express abnormal transcript variants from the NFE2L2 gene (encoding Nrf2) that lack exon 2, or exons 2 and 3, and encode Nrf2 protein isoforms missing the KEAP1 interaction domain. The Nrf2 alterations result in the loss of interaction with KEAP1, Nrf2 stabilization, induction of a Nrf2 transcriptional response, and Nrf2 pathway dependence. In all analyzed cases, transcript variants were the result of heterozygous genomic microdeletions. Thus, we identify an alternative mechanism for Nrf2 pathway activation in human tumors and elucidate its functional consequences. PMID:27568559

  16. A single nucleotide polymorphism in an exon dictates allele dependent differential splicing of episialin mRNA.

    PubMed Central

    Ligtenberg, M J; Gennissen, A M; Vos, H L; Hilkens, J

    1991-01-01

    The episialin gene (MUC1) encodes an epithelial mucin containing a variable number of repeats with a length of twenty amino acids, resulting in many different alleles that can be subdivided into two size classes. The episialin pre-mRNA uses either one of two neighbouring splice acceptor sites for exon 2, which mainly encodes the repeats. Using the genetic polymorphism of the episialin gene to identify different alleles, we show here that the splice site recognition is allele dependent and is based on a single A/G nucleotide difference in exon 2 eight nucleotides downstream of the second splice acceptor site. Transfection experiments confirm that this polymorphic nucleotide regulates the splice site selection. The identity of this nucleotide is in most cases correlated with one of the size classes of the alleles, indicating that mutations altering the number of repeats seldom arise by unequal cross-over between the repeat regions. Images PMID:2014168

  17. Targeted Exon Sequencing Successfully Discovers Rare Causative Genes and Clarifies the Molecular Epidemiology of Japanese Deafness Patients

    PubMed Central

    Kamatani, Naoyuki; Usami, Shin-ichi

    2013-01-01

    Target exon resequencing using Massively Parallel DNA Sequencing (MPS) is a new powerful strategy to discover causative genes in rare Mendelian disorders such as deafness. We attempted to identify genomic variations responsible for deafness by massive sequencing of the exons of 112 target candidate genes. By the analysis of 216randomly selected Japanese deafness patients (120 early-onset and 96 late-detected), who had already been evaluated for common genes/mutations by Invader assay and of which 48 had already been diagnosed, we efficiently identified causative mutations and/or mutation candidates in 57 genes. Approximately 86.6% (187/216) of the patients had at least one mutation. Of the 187 patients, in 69 the etiology of the hearing loss was completely explained. To determine which genes have the greatest impact on deafness etiology, the number of mutations was counted, showing that those in GJB2 were exceptionally higher, followed by mutations in SLC26A4, USH2A, GPR98, MYO15A, COL4A5 and CDH23. The present data suggested that targeted exon sequencing of selected genes using the MPS technology followed by the appropriate filtering algorithm will be able to identify rare responsible genes including new candidate genes for individual patients with deafness, and improve molecular diagnosis. In addition, using a large number of patients, the present study clarified the molecular epidemiology of deafness in Japanese. GJB2 is the most prevalent causative gene, and the major (commonly found) gene mutations cause 30–40% of deafness while the remainder of hearing loss is the result of various rare genes/mutations that have been difficult to diagnose by the conventional one-by-one approach. In conclusion, target exon resequencing using MPS technology is a suitable method to discover common and rare causative genes for a highly heterogeneous monogenic disease like hearing loss. PMID:23967202

  18. Exon-specific DNA hypomethylation of the p53 gene of rat colon induced by dimethylhydrazine. Modulation by dietary folate.

    PubMed Central

    Kim, Y. I.; Pogribny, I. P.; Salomon, R. N.; Choi, S. W.; Smith, D. E.; James, S. J.; Mason, J. B.

    1996-01-01

    Folate deficiency enhances colorectal carcinogenesis in dimethylhydrazine-treated rats. Folate is an important mediator of DNA methylation, an epigenetic modification of DNA that is known to be dysregulated in the early stages of colorectal cancer. This study investigated the effect of dimethylhydrazine on DNA methylation of the colonic p53 gene and the modulation of this effect by dietary folate. Sprague-Dawley rats were fed diets containing 0, 2, 8, or 40 mg of folate/kg of diet. Five weeks after diet initiation, dimethylhydrazine was injected weekly for fifteen weeks. Folate-depleted and folate-replete control animals did not receive dimethylhydrazine and were fed the 0- and 8-mg folate diets, respectively. The extent of p53 methylation was determined by a quantitative HpaII-polymerase chain reaction. In exons 6 and 7, significant p53 hypomethylation was observed in all dimethylhydrazine-treated rats relative to controls (P < 0.01), independent of dietary folate. In exon 8, significant p53 hypomethylation was observed only in the dimethylhydrazine-treated folate-depleted rats compared with controls (P = 0.038) and was effectively overcome by increasing levels of dietary folate (P = 0.008). In this model, dimethylhydrazine induces exon-specific p53 hypomethylation. In some exons, this occurs independent of dietary folate, and in others, increasing levels of dietary folate effectively override the induction of hypomethylation in a dose-responsive manner. This may be a mechanism by which increasing levels of dietary folate inhibit colorectal carcinogenesis. PMID:8863662

  19. Novel P2 promoter-derived HNF4{alpha} isoforms with different N-terminus generated by alternate exon insertion

    SciTech Connect

    Huang, Jianmin; Levitsky, Lynne L.; Rhoads, David B.

    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.

  20. Mutation analysis in exons 22 and 24 of SCN4A gene in Iranian patients with non-dystrophic myotonia

    PubMed Central

    Heidari, Mohammad Mehdi; Khatami, Mehri; Nafissi, Shahriar; Hesami-Zokai, Faezeh; Khorrami, Afshin

    2015-01-01

    Background: Non-dystrophic myotonias are a heterogeneous set of skeletal, muscular channelopathies, which have been associated with point mutations within sodium channel α-subunit (SCN4A) gene. Because exons 22 and 24 of SCN4A gene are recognized as hot spots for this disease, the purpose of the study is to identify mutation in exons 22 and 24 of SCN4A gene in Iranian non-dystrophic myotonias patients. Methods: In this study, 28 Iranian patients with non-dystrophic myotonia analyzed for the mutation scanning in exons 22 and 24 of SCN4A gene by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP) and sequencing. Results: We found 29073G>C substitution in SCN4A gene in one case and 31506A>G substitution in seven cases. The 29073G>C substitution causes a missense mutation G1306A, located in the conserved cytoplasmic loop connecting repeat III and IV of the SCN4A channel but, 31506A>G substitution do not alter amino acid in SCN4A protein. Conclusion: G1306A residue is located in functionally important protein region. In “hinged-lid model” for Na+ channel inactivation in which glycines1306 act as the hinge of the lid occluding the channel pore. Mutation in this region slowed fast inactivation. Therefore, it might be a pathogenic mutation. The causal relationship of this mutation with the disease is an object for further discussion. PMID:26885337

  1. Functional Classification of BRCA2 DNA Variants by Splicing Assays in a Large Minigene with 9 Exons

    PubMed Central

    Acedo, Alberto; Hernández-Moro, Cristina; Curiel-García, Álvaro; Díez-Gómez, Beatriz; Velasco, Eladio A

    2015-01-01

    Numerous pathogenic DNA variants impair the splicing mechanism in human genetic diseases. Minigenes are optimal approaches to test variants under the splicing viewpoint without the need of patient samples. We aimed to design a robust minigene construct of the breast cancer gene BRCA2 in order to investigate the impact of variants on splicing. BRCA2 exons 19–27 (MGBR2_ex19–27) were cloned in the new vector pSAD. It produced a large transcript of the expected size (2,174 nucleotides) and exon structure (V1-ex19-27-V2). Splicing assays showed that 18 (17 splice-site and 1 silencer variants) out of 40 candidate DNA variants induced aberrant patterns. Twenty-four anomalous transcripts were accurately detected by fluorescent-RT-PCR that were generated by exon-skipping, alternative site usage, and intron-retention events. Fourteen variants induced major anomalies and were predicted to disrupt protein function so they could be classified as pathogenic. Furthermore, minigene mimicked previously reported patient RNA outcomes of seven variants supporting the reproducibility of minigene assays. Therefore, a relevant fraction of variants are involved in breast cancer through splicing alterations. MGBR2_ex19–27 is the largest reported BRCA2 minigene and constitutes a valuable tool for the functional and clinical classification of sequence variations. PMID:25382762

  2. A region in the first exon/intron of rat carnitine palmitoyltransferase Ibeta is involved in enhancement of basal transcription.

    PubMed Central

    Wang, Guo-Li; Moore, Meredith L; McMillin, Jeanie B

    2002-01-01

    Carnitine palmitoyltransferase-Ibeta (CPT-Ibeta) catalyses the transfer of long-chain fatty acids to the enzymes of beta-oxidation of muscle and heart. Transcriptional control of this regulatory protein is relevant to disorders of fatty acid oxidation and the switch to glucose metabolism that occurs in cardiac pathology. The presence of a transcriptional enhancer sequence in the first untranslated exon and first intron of the CPT-Ibeta gene was identified using deletional and mutational analysis, and by ligation of an oleate responsive element (fatty acid response element) to a minimal promoter. The enhancer sequences are contained in the first 40 bases downstream of the transcription start site and increase CPT-Ibeta reporter gene expression independent of any 5' cis-acting elements. Deletion of the first 40 bases of the 3'-untranslated region does not affect the up-regulation of transcription by 10 microM phenylephrine. However, mutation and/or deletion of bases between +11 and +30 dramatically decreases reporter gene expression. Electrophoretic mobility-shift assays reveal two DNA (+11 to +36)-protein complexes that appear cardiac specific. The exon/intron element enhances activation of the heterologous thymidine kinase promoter in a position- and orientation-dependent manner. Therefore we have identified a novel region in the first exon/intron of the CPT-Ibeta gene that acts as a non-classical transcriptional enhancer downstream of regulatory elements characterized previously in the 5'-flanking region of the minimal promoter. PMID:11879187

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

    PubMed

    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. PMID:24320790

  4. Human Tra2 proteins jointly control a CHEK1 splicing switch among alternative and constitutive target exons

    PubMed Central

    Best, Andrew; James, Katherine; Dalgliesh, Caroline; Hong, Elaine; Kheirolahi-Kouhestani, Mahsa; Curk, Tomaz; Xu, Yaobo; Danilenko, Marina; Hussain, Rafiq; Keavney, Bernard; Wipat, Anil; Klinck, Roscoe; Cowell, Ian G.; Cheong Lee, Ka; Austin, Caroline A.; Venables, Julian P.; Chabot, Benoit; Santibanez Koref, Mauro; Tyson-Capper, Alison; Elliott, David J.

    2014-01-01

    Alternative splicing—the production of multiple messenger RNA isoforms from a single gene—is regulated in part by RNA binding proteins. While the RBPs transformer2 alpha (Tra2α) and Tra2β have both been implicated in the regulation of alternative splicing, their relative contributions to this process are not well understood. Here we find simultaneous—but not individual—depletion of Tra2α and Tra2β induces substantial shifts in splicing of endogenous Tra2β target exons, and that both constitutive and alternative target exons are under dual Tra2α–Tra2β control. Target exons are enriched in genes associated with chromosome biology including CHEK1, which encodes a key DNA damage response protein. Dual Tra2 protein depletion reduces expression of full-length CHK1 protein, results in the accumulation of the DNA damage marker γH2AX and decreased cell viability. We conclude Tra2 proteins jointly control constitutive and alternative splicing patterns via paralog compensation to control pathways essential to the maintenance of cell viability. PMID:25208576

  5. Alternative exon 9-encoded relay domains affect more than one communication pathway in the Drosophila myosin head.

    PubMed

    Bloemink, Marieke J; Dambacher, Corey M; Knowles, Aileen F; Melkani, Girish C; Geeves, Michael A; Bernstein, Sanford I

    2009-06-19

    We investigated the biochemical and biophysical properties of one of the four alternative regions within the Drosophila