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‣ Rare Autosomal Recessive Cardiac Valvular Form of Ehlers-Danlos Syndrome Results from Mutations in the COL1A2 Gene That Activate the Nonsense-Mediated RNA Decay Pathway

Schwarze, Ulrike; Hata, Ryu-Ichiro; McKusick, Victor A.; Shinkai, Hiroshi; Hoyme, H. Eugene; Pyeritz, Reed E.; Byers, Peter H.
Fonte: The American Society of Human Genetics Publicador: The American Society of Human Genetics
Tipo: Artigo de Revista Científica
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Splice site mutations in the COL1A2 gene of type I collagen can give rise to forms of Ehlers-Danlos syndrome (EDS) because of partial or complete skipping of exon 6, as well as to mild, moderate, or lethal forms of osteogenesis imperfecta as a consequence of skipping of other exons. We identified three unrelated individuals with a rare recessively inherited form of EDS (characterized by joint hypermobility, skin hyperextensibility, and cardiac valvular defects); in two of them, COL1A2 messenger RNA (mRNA) instability results from compound heterozygosity for splice site mutations in the COL1A2 gene, and, in the third, it results from homozygosity for a nonsense codon. The splice site mutations led to use of cryptic splice donor sites, creation of a downstream premature termination codon, and extremely unstable mRNA. In the wild-type allele, the two introns (IVS11 and IVS24) in which these mutations occurred were usually spliced slowly in relation to their respective immediate upstream introns. In the mutant alleles, the upstream intron was removed, so that exon skipping could not occur. In the context of the mutation in IVS24, computer-generated folding of a short stretch of mRNA surrounding the mutation site demonstrated realignment of the relationships between the donor and acceptor sites that could facilitate use of a cryptic donor site. These findings suggest that the order of intron removal is an important variable in prediction of mutation outcome at splice sites and that folding of the nascent mRNA could be one element that contributes to determination of order of splicing. The complete absence of proα2(I) chains has the surprising effect of producing cardiac valvular disease without bone involvement.

‣ Complex splicing control of the human Thrombopoietin gene by intronic G runs

Marcucci, Roberto; Baralle, Francisco E.; Romano, Maurizio
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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The human thrombopoietin (THPO) gene displays a series of alternative splicing events that provide valuable models for studying splicing mechanisms. The THPO region spanning exon 1–4 presents both alternative splicing of exon 2 and partial intron 2 (IVS2) retention following the activation of a cryptic 3′ splice site 85 nt upstream of the authentic acceptor site. IVS2 is particularly rich in stretches of 3–5 guanosines (namely, G1–G10) and we have characterized the role of these elements in the processing of this intron. In vivo studies show that runs G7–G10 work in a combinatorial way to control the selection of the proper 3′ splice site. In particular, the G7 element behaves as the splicing hub of intron 2 and its interaction with hnRNP H1 is critical for the splicing process. Removal of hnRNP H1 by RNA interference promoted the usage of the cryptic 3′ splice site so providing functional evidence that this factor is involved in the selection of the authentic 3′ splice site of THPO IVS2.

‣ Cryptic Exon Activation by Disruption of Exon Splice Enhancer: NOVEL MECHANISM CAUSING 3-METHYLCROTONYL-CoA CARBOXYLASE DEFICIENCY*

Stucki, Martin; Suormala, Terttu; Fowler, Brian; Valle, David; Baumgartner, Matthias R.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
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3-Methylcrotonyl-CoA carboxylase (MCC) deficiency is an autosomal recessive disorder of leucine catabolism. MCC is a heteromeric mitochondrial enzyme composed of biotin-containing α (MCCA) and smaller β (MCCB) subunits encoded by MCCA and MCCB, respectively. We report studies of the c.1054G→A mutation in exon 11 of MCCB detected in the homozygous state in a patient with MCC deficiency. Sequence analysis of MCCB cDNA revealed two overlapping transcripts, one containing the normal 73 bp of exon 11 including the missense mutation c.1054G→A (p.G352R), the other with exon 11 replaced by a 64-bp sequence from intron 10 (cryptic exon 10a) that maintains the reading frame and is flanked by acceptable splice consensus sites. In expression studies, we show that both transcripts lack detectable MCC activity. Western blot analysis showed slightly reduced levels of MCCB using the transcript containing the missense mutation, whereas no MCCB was detected with the transcript containing the cryptic exon 10a. Analysis of the region harboring the mutation revealed that the c.1054G→A mutation is located in an exon splice enhancer sequence. Using MCCB minigene constructs to transfect MCCB-deficient fibroblasts, we demonstrate that the reduction in utilization of exon 11 associated with the c.1054G→A mutation is due to alteration of this exon splice enhancer. Further...

‣ Analysis of novel sph (spherocytosis) alleles in mice reveals allele-specific loss of band 3 and adducin in α-spectrin–deficient red cells

Robledo, Raymond F.; Lambert, Amy J.; Birkenmeier, Connie S.; Cirlan, Marius V.; Cirlan, Andreea Flavia M.; Campagna, Dean R.; Lux, Samuel E.; Peters, Luanne L.
Fonte: American Society of Hematology Publicador: American Society of Hematology
Tipo: Artigo de Revista Científica
Publicado em 04/03/2010 Português
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Five spontaneous, allelic mutations in the α-spectrin gene, Spna1, have been identified in mice (spherocytosis [sph], sph1J, sph2J, sph2BC, sphDem). All cause severe hemolytic anemia. Here, analysis of 3 new alleles reveals previously unknown consequences of red blood cell (RBC) spectrin deficiency. In sph3J, a missense mutation (H2012Y) in repeat 19 introduces a cryptic splice site resulting in premature termination of translation. In sphIhj, a premature stop codon occurs (Q1853Stop) in repeat 18. Both mutations result in markedly reduced RBC membrane spectrin content, decreased band 3, and absent β-adducin. Reevaluation of available, previously described sph alleles reveals band 3 and adducin deficiency as well. In sph4J, a missense mutation occurs in the C-terminal EF hand domain (C2384Y). Notably, an equally severe hemolytic anemia occurs despite minimally decreased membrane spectrin with normal band 3 levels and present, although reduced, β-adducin. The severity of anemia in sph4J indicates that the highly conserved cysteine residue at the C-terminus of α-spectrin participates in interactions critical to membrane stability. The data reinforce the notion that a membrane bridge in addition to the classic protein 4.1-p55-glycophorin C linkage exists at the RBC junctional complex that involves interactions between spectrin...

‣ Spliceosome discards intermediates via the DEAH box ATPase Prp43p

Mayas, Rabiah M.; Maita, Hiroshi; Semlow, Daniel R.; Staley, Jonathan P.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
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To promote fidelity in nuclear pre-mRNA splicing, the spliceosome rejects and discards suboptimal substrates that have engaged the spliceosome. Whereas DExD/H box ATPases have been implicated in rejecting suboptimal substrates, the mechanism for discarding suboptimal substrates has remained obscure. Corroborating evidence that suboptimal, mutated lariat intermediates can be exported to the cytoplasm for turnover, we have found that the ribosome can translate mutated lariat intermediates. By glycerol gradient analysis, we have found that the spliceosome can dissociate mutated lariat intermediates in vivo in a manner that requires the DEAH box ATPase Prp43p. Through an in vitro assay, we demonstrate that Prp43p promotes the discard of suboptimal and optimal 5′ exon and lariat intermediates indiscriminately. Finally, we demonstrate a requirement for Prp43p in repressing splicing at a cryptic splice site. We propose a model for the fidelity of exon ligation in which the DEAH box ATPase Prp22p slows the flow of suboptimal intermediates through exon ligation and Prp43p generally promotes discard of intermediates, thereby establishing a pathway for turnover of stalled intermediates. Because Prp43p also promotes spliceosome disassembly after exon ligation...

‣ Terminal Osseous Dysplasia Is Caused by a Single Recurrent Mutation in the FLNA Gene

Sun, Yu; Almomani, Rowida; Aten, Emmelien; Celli, Jacopo; van der Heijden, Jaap; Venselaar, Hanka; Robertson, Stephen P.; Baroncini, Anna; Franco, Brunella; Basel-Vanagaite, Lina; Horii, Emiko; Drut, Ricardo; Ariyurek, Yavuz; den Dunnen, Johan T.; Breunin
Fonte: Elsevier Publicador: Elsevier
Tipo: Artigo de Revista Científica
Publicado em 09/07/2010 Português
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Terminal osseous dysplasia (TOD) is an X-linked dominant male-lethal disease characterized by skeletal dysplasia of the limbs, pigmentary defects of the skin, and recurrent digital fibroma with onset in female infancy. After performing X-exome capture and sequencing, we identified a mutation at the last nucleotide of exon 31 of the FLNA gene as the most likely cause of the disease. The variant c.5217G>A was found in six unrelated cases (three families and three sporadic cases) and was not found in 400 control X chromosomes, pilot data from the 1000 Genomes Project, or the FLNA gene variant database. In the families, the variant segregated with the disease, and it was transmitted four times from a mildly affected mother to a more seriously affected daughter. We show that, because of nonrandom X chromosome inactivation, the mutant allele was not expressed in patient fibroblasts. RNA expression of the mutant allele was detected only in cultured fibroma cells obtained from 15-year-old surgically removed material. The variant activates a cryptic splice site, removing the last 48 nucleotides from exon 31. At the protein level, this results in a loss of 16 amino acids (p.Val1724_Thr1739del), predicted to remove a sequence at the surface of filamin repeat 15. Our data show that TOD is caused by this single recurrent mutation in the FLNA gene.

‣ Deafness and Retinal Degeneration in A Novel USH1C Knock-In Mouse Model

Lentz, Jennifer J.; Gordon, William C.; Farris, Hamilton E.; MacDonald, Glen H.; Cunningham, Dale E.; Robbins, Carol A.; Tempel, Bruce L.; Bazan, Nicolas G.; Rubel, Edwin W.; Oesterle, Elizabeth C.; Keats, Bronya J.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /03/2010 Português
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Usher syndrome is the leading cause of combined deaf-blindness, but the molecular mechanisms underlying the auditory and visual impairment are poorly understood. Usher I is characterized by profound congenital hearing loss, vestibular dysfunction and progressive retinitis pigmentosa beginning in early adolescence. Using the c.216G>A cryptic splice site mutation in exon 3 of the USH1C gene found in Acadian Usher I patients in Louisiana, we constructed the first mouse model that develops both deafness and retinal degeneration. The same truncated mRNA transcript found in Usher 1C patients is found in the cochleae and retinas of these knock-in mice. Absent auditory-evoked brainstem responses indicated that the mutant mice are deaf at one month of age. Cochlear histology showed disorganized hair cell rows, abnormal bundles, and loss of both inner and outer hair cells in the middle turns and at the base. Retinal dysfunction as evident by an abnormal electroretinogram was seen as early as 1 month of age, with progressive loss of rod photoreceptors between 6 and 12 months of age. This knock-in mouse reproduces the dual sensory loss of human Usher I, providing a novel resource to study the disease mechanism and the development of therapies.

‣ Importance of molecular cell biology investigations in human medicine in the story of the Hutchinson-Gilford progeria syndrome

Raška, Ivan
Fonte: Slovak Toxicology Society SETOX Publicador: Slovak Toxicology Society SETOX
Tipo: Artigo de Revista Científica
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Ranged among laminopathies, Hutchinson–Gilford progeria syndrome is a syndrome that involves premature aging, leading usually to death at the age between 10 to 14 years predominatly due to a myocardial infarction or a stroke. In the lecture I shall overview the importance of molecular cell biology investigations that led to the discovery of the basic mechanism standing behind this rare syndrome. The genetic basis in most cases is a mutation at the nucleotide position 1824 of the lamin A gene. At this position, cytosine is substituted for thymine so that a cryptic splice site within the precursor mRNA for lamin A is generated. This results in a production of abnormal lamin A, termed progerin, its presence in cells having a deleterious dominant effect. Depending on the cell type and tissue, progerin induces a pleiotropy of defects that vary in different tissues. The present endeavour how to challenge this terrible disease will be also mentioned.

‣ Combining fetal sonography with genetic and allele pathogenicity studies to secure a neonatal diagnosis of Bardet–Biedl syndrome

Ashkinadze, E.; Rosen, T.; Brooks, SS.; Katsanis, N.; Davis, EE.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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Bardet–Biedl syndrome (BBS) is a rare pediatric ciliopathy characterized by marked clinical variability and extensive genetic heterogeneity. Typical diagnosis of BBS is secured at a median of 9 years of age, and sometimes well into adolescence. Here, we report a patient in whom prenatal detection of increased nuchal fold, enlarged echogenic kidneys, and polydactyly prompted us to screen the most commonly mutated genes in BBS and the phenotypically and genetically overlapping ciliopathy, Meckel–Gruber syndrome (MKS). We identified the common Met390Arg mutation in BBS1 in compound heterozygosity with a novel intronic variant of unknown significance (VUS). Testing of mRNA harvested from primary foreskin fibroblasts obtained shortly after birth revealed the VUS to induce a cryptic splice site, which in turn led to a premature termination and mRNA degradation. To our knowledge, this is the earliest diagnosis of BBS in the absence of other affected individuals in the family, and exemplifies how combining clinical assessment with genetic and timely assays of variant pathogenicity can inform clinical diagnosis and assist with patient management in the prenatal and neonatal setting.

‣ Negative Feedback Control of Jasmonate Signaling by an Alternative Splice Variant of JAZ101[C][W][OA]

Moreno, Javier E.; Shyu, Christine; Campos, Marcelo L.; Patel, Lalita C.; Chung, Hoo Sun; Yao, Jian; He, Sheng Yang; Howe, Gregg A.
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica
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A transcriptional regulator generated by alternative splicing uses a cryptic binding site to negatively regulate bHLH-type transcription factors that promote jasmonate responses.

‣ Ethnic-specific WRN mutations in South Asian Werner syndrome patients: potential founder effect in patients with Indian or Pakistani ancestry

Saha, Bidisha; Lessel, Davor; Nampoothiri, Sheela; Rao, Anuradha S; Hisama, Fuki M; Peter, Dincy; Bennett, Chris; Nürnberg, Gudrun; Nürnberg, Peter; Martin, George M; Kubisch, Christian; Oshima, Junko
Fonte: Blackwell Publishing Ltd Publicador: Blackwell Publishing Ltd
Tipo: Artigo de Revista Científica
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Werner syndrome (WS) is a rare autosomal recessive disorder characterized by multiple features consistent with accelerated aging. It is caused by mutations in the WRN gene, which encodes a RecQ type helicase. To date, more than 70 disease-causing mutations have been reported. While founder mutations and a corresponding relatively high incidence of WS have been reported in Japan and Sardinia, such mutations have not been previously described among patients of South Asian descent. Here, we report two novel WRN mutations in three pedigrees. A homozygous c.561A>G mutation in exon 6 was identified both in a pedigree from Kerala, India and in a British patient of Pakistani ancestry. Although c.561A>G does not alter the corresponding amino acid (p.Lys187), it creates a cryptic splice site resulting in a 98 bp deletion at the mRNA level (r.557_654del98) followed by a frameshift (p.Lys187Trpfs*13). These two cases shared the same haplotype across the WRN gene, and were distinct from another Indian Werner patient with a homozygous stop codon mutation, c.2855 C > A (p.Ser952*), in exon 24. As the Indian population increases and the awareness of WS grows, we anticipate that more cases will be identified with these founder mutations among South Asian WS patients.

‣ Validation of predicted mRNA splicing mutations using high-throughput transcriptome data

Viner, Coby; Dorman, Stephanie N.; Shirley, Ben C.; Rogan, Peter K.
Fonte: F1000Research Publicador: F1000Research
Tipo: Artigo de Revista Científica
Publicado em 07/04/2014 Português
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Interpretation of variants present in complete genomes or exomes reveals numerous sequence changes, only a fraction of which are likely to be pathogenic. Mutations have been traditionally inferred from allele frequencies and inheritance patterns in such data. Variants predicted to alter mRNA splicing can be validated by manual inspection of transcriptome sequencing data, however this approach is intractable for large datasets. These abnormal mRNA splicing patterns are characterized by reads demonstrating either exon skipping, cryptic splice site use, and high levels of intron inclusion, or combinations of these properties. We present, Veridical, an in silico method for the automatic validation of DNA sequencing variants that alter mRNA splicing. Veridical performs statistically valid comparisons of the normalized read counts of abnormal RNA species in mutant versus non-mutant tissues. This leverages large numbers of control samples to corroborate the consequences of predicted splicing variants in complete genomes and exomes.

‣ Clinical, Biochemical, and Molecular Characterization of Novel Mutations in ABCA1 in Families with Tangier Disease

Brunham, Liam R.; Kang, Martin H.; Van Karnebeek, Clara; Sadananda, Singh N.; Collins, Jennifer A.; Zhang, Lin-Hua; Sayson, Bryan; Miao, Fudan; Stockler, Sylvia; Frohlich, Jiri; Cassiman, David; Rabkin, Simon W.; Hayden, Michael R.
Fonte: Springer Berlin Heidelberg Publicador: Springer Berlin Heidelberg
Tipo: Artigo de Revista Científica
Publicado em 12/10/2014 Português
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Tangier disease is a rare, autosomal recessive disorder caused by mutations in the ABCA1 gene and is characterized by near absence of plasma high-density lipoprotein cholesterol, accumulation of cholesterol in multiple tissues, peripheral neuropathy, and accelerated atherosclerosis. Here we report three new kindreds with Tangier disease harboring both known and novel mutations in ABCA1. One patient was identified to be homozygous for a nonsense mutation, p.Gln1038*. In a remarkably large Tangier disease pedigree with four affected siblings, we identified compound heterozygosity for previously reported missense variants, p.Arg937Val and p.Thr940Met, and show that both of these mutations result in significantly impaired cholesterol efflux in transfected cells. In a third pedigree, the proband was identified to be compound heterozygous for two novel mutations, a frameshift (p.Ile1200Hisfs*4) and an intronic variant (c.4176-11T>G), that lead to the creation of a cryptic splice site acceptor and premature truncation, p.Ser1392Argfs*6. We demonstrate that this mutation arose de novo, the first demonstration of a pathogenic de novo mutation in ABCA1 associated with Tangier disease. We also report results of glucose tolerance testing in a Tangier disease kindred for the first time...

‣ Interlocked circle formation by group I introns: structural requirements and mechanism.

Winter, A J; Alkema, M J; Groot Koerkamp, M J; van der Horst, G; Mul, Y; Tabak, H F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/07/1993 Português
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Precursor RNA transcribed from the yeast mitochondrial gene coding for the large ribosomal RNA contains a group I intron that can excise itself in vitro. Apart from group I specific sequence elements the intron also contains a gene encoding a DNA endonuclease involved in intron dispersal. A precursor RNA derivative from which this gene has been removed self-splices efficiently, but due to activation of cryptic opening sites located in the 5' exon, the 3' part of this exon is sometimes co-excised with the intron. Upon further reaction, this enlarged intron molecules give rise to interlocked circles, comprising small circles derived from 5' exon parts and large circles of the intron. Sequence comparison between cryptic opening sites and authentic splice sites reveals in most cases homology with the 3' exon part that is capable of interacting with the Internal Guide Sequence. The role of the IGS was further substantiated by replacing the cryptic opening sites with well defined sequences of authentic splice sites: one corresponding to the 3' splice site and its mutant derivatives, the other to a fragment containing the natural 5'-3' exon junction. Precursor RNAs derived from these constructs give rise to interlocked circles, and mutation studies confirm that the 3' exon nucleotides flanking a 3' splice site are essential for their formation. The results underline the crucial role of the IGS in interlocked circle formation which behaves similarly as in the normal self-splicing reactions. It has been proposed that the two short helices formed by basepairing of the IGS with the 5' and 3' exon can co-axially stack on top of each other forming a quasi continuous RNA double helix or pseudoknot. We present a model explaining how transesterification reactions of a mutant precursor RNA in such a pseudoknot can lead to interlocked circles. The experiments support the notion that a similar structure is also operative in splicing of wild type precursor RNA.

‣ Neonatal progeria: increased ratio of progerin to lamin A leads to progeria of the newborn

Reunert, Janine; Wentzell, Rüdiger; Walter, Michael; Jakubiczka, Sibylle; Zenker, Martin; Brune, Thomas; Rust, Stephan; Marquardt, Thorsten
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
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Hutchinson–Gilford progeria syndrome (HGPS) is an important model disease for premature ageing. Affected children appear healthy at birth, but develop the first symptoms during their first year of life. They die at an average age of 13 years, mostly because of myocardial infarction or stroke. Classical progeria is caused by the heterozygous point mutation c.1824C>T in the LMNA gene, which activates a cryptic splice site. The affected protein cannot be processed correctly to mature lamin A, but is modified into a farnesylated protein truncated by 50 amino acids (progerin). Three more variations in LMNA result in the same mutant protein, but different grades of disease severity. We describe a patient with the heterozygous LMNA mutation c.1821G>A, leading to neonatal progeria with death in the first year of life. Intracellular lamin A was downregulated in the patient's fibroblasts and the ratio of progerin to lamin A was increased when compared with HGPS. It is suggestive that the ratio of farnesylated protein to mature lamin A determines the disease severity in progeria.

‣ Exploitation of a thermosensitive splicing event to study pre-mRNA splicing in vivo.

Cizdziel, P E; de Mars, M; Murphy, E C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1988 Português
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The spliced form of MuSVts110 viral RNA is approximately 20-fold more abundant at growth temperatures of 33 degrees C or lower than at 37 to 41 degrees C. This difference is due to changes in the efficiency of MuSVts110 RNA splicing rather than selective thermolability of the spliced species at 37 to 41 degrees C or general thermosensitivity of RNA splicing in MuSVts110-infected cells. Moreover, RNA transcribed from MuSVts110 DNA introduced into a variety of cell lines is spliced in a temperature-sensitive fashion, suggesting that the structure of the viral RNA controls the efficiency of the event. We exploited this novel splicing event to study the cleavage and ligation events during splicing in vivo. No spliced viral mRNA or splicing intermediates were observed in MuSVts110-infected cells (6m2 cells) at 39 degrees C. However, after a short (about 30-min) lag following a shift to 33 degrees C, viral pre-mRNA cleaved at the 5' splice site began to accumulate. Ligated exons were not detected until about 60 min following the initial detection of cleavage at the 5' splice site, suggesting that these two splicing reactions did not occur concurrently. Splicing of viral RNA in the MuSVts110 revertant 54-5A4, which lacks the sequence -AG/TGT- at the usual 3' splice site...

‣ Molecular analysis of the androgen-receptor gene in a family with receptor-positive partial androgen insensitivity: an unusual type of intronic mutation.

Brüggenwirth, H T; Boehmer, A L; Ramnarain, S; Verleun-Mooijman, M C; Satijn, D P; Trapman, J; Grootegoed, J A; Brinkmann, A O
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /11/1997 Português
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In the coding part and the intron-exon boundaries of the androgen-receptor gene of a patient with partial androgen insensitivity, no mutation was found. The androgen receptor of this patient displayed normal ligand-binding parameters and migrated as a 110-112-kD doublet on SDS-PAGE in the absence of hormone. However, after culturing of the patient's genital skin fibroblasts in the presence of hormone, the slower-migrating 114-kD protein, which reflects hormone-dependent phosphorylation, was hardly detectable. Furthermore, receptor protein was undetectable in the nuclear fraction of the fibroblasts, after treatment with hormone, which is indicative of defective DNA binding. By sequencing part of intron 2, a T-->A mutation was found 11 bp upstream of exon 3. In our screening of 102 chromosomes from unrelated individuals, this base-pair substitution was not found, indicating that it was not a polymorphism. mRNA analysis revealed that splicing involved a cryptic splice site, located 71/70 bp upstream of exon 3, resulting in generation of mRNA with an insert of 69 nucleotides. In addition, a small amount of a transcript with a deleted exon 3 and a very low level of wild-type transcript were detected. Translation of the extended transcript resulted in an androgen-receptor protein with 23 amino acid residues inserted between the two zinc clusters...

‣ Trans-splicing and alternative-tandem-cis-splicing: two ways by which mammalian cells generate a truncated SV40 T-antigen.

Eul, J; Graessmann, M; Graessmann, A
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/05/1996 Português
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The early SV40 BstXI-BamHI (Bst/Bam) DNA fragment encodes exclusively for the second exon of the large T-antigen and contains the intact small t-antigen intron. Rat cells transformed by the p14T, a construct that carries the Bst/Bam DNA fragment as a tail-to-head tandem duplication, synthesize a truncated T-antigen (T1-antigen) without having a direct equivalent at the DNA level. Formation of the T1-mRNA occurs by means of two distinct mechanisms: alternative-tandem-cis-splicing and trans-splicing. To generate the T1-mRNA the cells utilize a cryptic 5' splice site, located within the second exon of the large T-antigen and the regular small t-antigen 3' splice site. Since these splice sites are in an inverted order two Bst/Bam transcripts are required to generate one T1-mRNA molecule. For alternative-tandem-cis-splicing the cells utilize a 4.4 kb pre-mRNA that contains the sequence of the entire Bst/Bam tandem repeat. The proximal Bst/Bam segment provides the 5' donor splice site and the distal segment the 3' acceptor site. This requires that the pre-mRNA not be cleaved after the RNA polymerase II has passed the polyadenylation signal of the proximal Bst/Bam DNA segment. Synthesis of the 4.4 kb pre-mRNA was demonstrable by RT-PCR but not by Northern blot analysis. For trans-splicing...

‣ A novel Alu-like element rearranged in the dystrophin gene causes a splicing mutation in a family with X-linked dilated cardiomyopathy.

Ferlini, A; Galié, N; Merlini, L; Sewry, C; Branzi, A; Muntoni, F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1998 Português
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We have identified and characterized a genomic sequence with some features typical of Alu-like mobile elements rearranged into the dystrophin gene in a family affected by X-linked dilated cardiomyopathy. The Alu-like sequence rearrangement occurred 2.4 kb downstream from the 5' end of intron 11 of the dystrophin gene. This rearrangement activated one cryptic splice site in intron 11 and produced an alternative transcript containing the Alu-like sequence and part of the adjacent intron 11, spliced between exons 11 and 12. Translation of this alternative transcript is truncated because of the numerous stop codons present in every frame of the Alu-like sequence. Only the mutant mRNA was detected in the heart muscle, but in the skeletal muscle it coexisted with the normal one. This result is supported by the immunocytochemical findings, which failed to detect dystrophin in the patient's cardiac muscle but showed expression of a reduced level of protein in the skeletal muscle. Comparative analysis of the Alu-like sequence showed high homology with other repeated-element-containing regions and with several expressed sequence tags. We suggest that this Alu-like sequence could represent a novel class of repetitive elements, reiterated and clustered with some known mobile elements and capable of transposition. Our report underlines the complexity of the pathogenic mechanism leading to X-linked dilated cardiomyopathy but suggests that differences in tissue-specific expression of dystrophin mutations may be a common feature in this condition.

‣ Identification and Characterization of Novel Variations in Platelet G-Protein Coupled Receptor (GPCR) Genes in Patients Historically Diagnosed with Type 1 von Willebrand Disease

Stockley, Jacqueline; Nisar, Shaista P.; Leo, Vincenzo C.; Sabi, Essa; Cunningham, Margaret R.; Eikenboom, Jeroen C.; Lethagen, Stefan; Schneppenheim, Reinhard; Goodeve, Anne C.; Watson, Steve P.; Mundell, Stuart J.; Daly, Martina E.;
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 02/12/2015 Português
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The clinical expression of type 1 von Willebrand disease may be modified by co-inheritance of other mild bleeding diatheses. We previously showed that mutations in the platelet P2Y12 ADP receptor gene (P2RY12) could contribute to the bleeding phenotype in patients with type 1 von Willebrand disease. Here we investigated whether variations in platelet G protein-coupled receptor genes other than P2RY12 also contributed to the bleeding phenotype. Platelet G protein-coupled receptor genes P2RY1, F2R, F2RL3, TBXA2R and PTGIR were sequenced in 146 index cases with type 1 von Willebrand disease and the potential effects of identified single nucleotide variations were assessed using in silico methods and heterologous expression analysis. Seven heterozygous single nucleotide variations were identified in 8 index cases. Two single nucleotide variations were detected in F2R; a novel c.-67G>C transversion which reduced F2R transcriptional activity and a rare c.1063C>T transition predicting a p.L355F substitution which did not interfere with PAR1 expression or signalling. Two synonymous single nucleotide variations were identified in F2RL3 (c.402C>G, p.A134 =; c.1029 G>C p.V343 =), both of which introduced less commonly used codons and were predicted to be deleterious...