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‣ Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome

LIMA, Renata. L. L. Ferreira de; HOPER, Sarah A.; GHASSIBE, Michella; COOPER, Margaret E.; RORICK, Nicholas K.; KONDO, Shinji; KATZ, Lori; MARAZITA, Mary L.; COMPTON, John; BALE, Sherri; HEHR, Ute; DIXON, Michael J.; DAACK-HIRSCH, Sandra; BOUTE, Odile; BA
Fonte: LIPPINCOTT WILLIAMS & WILKINS Publicador: LIPPINCOTT WILLIAMS & WILKINS
Tipo: Artigo de Revista Científica
Português
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Purpose: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic Mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. Methods: We performed direct sequence analysis of interferon regulatory factor 6 exons oil samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. Results: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further...

‣ Prevalence and nonrandom distribution of exonic mutations in interferon regulatory factor 6 in 307 families with Van der Woude syndrome and 37 families with popliteal pterygium syndrome

Lima, Renata. L. L. Ferreira de; Hoper, Sarah A.; Ghassibe, Michella; Cooper, Margaret E.; Rorick, Nicholas K.; Kondo, Shinji; Katz, Lori; Marazita, Mary L.; Compton, John; Bale, Sherri; Hehr, Ute; Dixon, Michael J.; Daack-Hirsch, Sandra; Boute, Odile; Ba
Fonte: Lippincott Williams & Wilkins Publicador: Lippincott Williams & Wilkins
Tipo: Artigo de Revista Científica Formato: 241-247
Português
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Purpose: Interferon regulatory factor 6 encodes a member of the IRF family of transcription factors. Mutations in interferon regulatory factor 6 cause Van der Woude and popliteal pterygium syndrome, two related orofacial clefting disorders. Here, we compared and contrasted the frequency and distribution of exonic Mutations in interferon regulatory factor 6 between two large geographically distinct collections of families with Van der Woude and between one collection of families with popliteal pterygium syndrome. Methods: We performed direct sequence analysis of interferon regulatory factor 6 exons oil samples from three collections, two with Van der Woude and one with popliteal pterygium syndrome. Results: We identified mutations in interferon regulatory factor 6 exons in 68% of families in both Van der Woude collections and in 97% of families with popliteal pterygium syndrome. In sum, 106 novel disease-causing variants were found. The distribution of mutations in the interferon regulatory factor 6 exons in each collection was not random; exons 3, 4, 7, and 9 accounted for 80%. In the Van der Woude collections, the mutations were evenly divided between protein truncation and missense, whereas most mutations identified in the popliteal pterygium syndrome collection were missense. Further...

‣ Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells.

Reichel, C; Mathur, J; Eckes, P; Langenkemper, K; Koncz, C; Schell, J; Reiss, B; Maas, C
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 11/06/1996 Português
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The expression of the jellyfish green fluorescent protein (GFP) in plants was analyzed by transient expression in protoplasts from Nicotiana tabacum, Arabidopsis thaliana, Hordeum vulgare, and Zea mays. Expression of GFP was only observed with a mutated cDNA, from which a recently described cryptic splice site had been removed. However, detectable levels of green fluorescence were only emitted from a small number of protoplasts. Therefore, other mutations in the GFP cDNA leading to single-amino acid exchanges in the chromophore region, which had been previously studied in Escherichia coli, were tested in order to improve the sensitivity of this marker protein. Of the mutations tested so far, the exchange of GFP amino acid tyrosine 66 to histidine (Y66H) led to detection of blue fluorescence in plant protoplasts, while the exchange of amino acid serine 65 to cysteine (S65C) and threonine (S65T) increased the intensity of green fluorescence drastically, thereby significantly raising the detection level for GFP. For GFP S65C, the detectable number of green fluorescing tobacco (BY-2) protoplasts was raised up to 19-fold, while the fluorimetricly determined fluorescence was raised by at least 2 orders of magnitude.

‣ Antisense properties of tricyclo-DNA

Renneberg, Dorte; Bouliong, Emilie; Reber, Ulrich; Schümperli, Daniel; Leumann, Christian J.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 01/07/2002 Português
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Tricyclo (tc)-DNA belongs to the class of conformationally constrained DNA analogs that show enhanced binding properties to DNA and RNA. We prepared tc-oligonucleotides up to 17 nt in length, and evaluated their binding efficiency and selectivity towards complementary RNA, their biological stability in serum, their RNase H inducing potential and their antisense activity in a cellular assay. Relative to RNA or 2′-O-Me-phosphorothioate (PS)-RNA, fully modified tc-oligodeoxynucleotides, 10–17 nt in length, show enhanced selectivity and enhanced thermal stability by ∼1°C/modification in binding to RNA targets. Tricyclodeoxyoligonucleotides are completely stable in heat-deactivated fetal calf serum at 37°C. Moreover, tc-DNA–RNA duplexes are not substrates for RNase H. To test for antisense effects in vivo, we used HeLa cell lines stably expressing the human β-globin gene with two different point mutations in the second intron. These mutations lead to the inclusion of an aberrant exon in β-globin mRNA. Lipofectamine-mediated delivery of a 17mer tc-oligodeoxynucleotide complementary to the 3′-cryptic splice site results in correction of aberrant splicing already at nanomolar concentrations with up to 100-fold enhanced efficiency relative to a 2′-O-Me-PS-RNA oligonucleotide of the same length and sequence. In contrast to 2′-O-Me-PS-RNA...

‣ Construction and functional characterization of polyomavirus genomes that separately encode the three early proteins.

Zhu, Z Y; Veldman, G M; Cowie, A; Carr, A; Schaffhausen, B; Kamen, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1984 Português
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Modified polyomavirus genomes that individually encode the large and small T proteins were constructed by exchanging restriction endonuclease fragments between cDNA copies of the respective mRNAs and cloned genomic DNA. The efficacies of the new constructs, and that of the middle T protein gene described previously (R. Treisman , U. Novak, J. Favaloro , and R. Kamen , Nature [London] 292:595-600, 1981), were demonstrated with simian virus 40 (SV40)-polyomavirus recombinants in which part or all of the SV40 late region was replaced with the modified polyomavirus early genes. Each of the three recombinant viruses induced the synthesis of only the expected polyomavirus early protein in infected CV-1 cells. The rates of synthesis of large, middle, and small T proteins were ca. 1.5, 4.0, and 9.0 times the rate of synthesis of SV40 large T protein, respectively. The deletion of introns had no detrimental effect on mRNA biogenesis. Indeed, a further polyomavirus-SV40 recombinant, containing wild-type polyomavirus early region DNA, expressed an aberrant 58,000-dalton form of the middle T protein which we believe to result from utilization of a cryptic splice site. Immunofluorescence studied with monkey cells infected by the recombinant viruses allowed us to determine the cellular locations of the polyomavirus early proteins. Overproduction of the middle T protein did not result in a corresponding overproduction of the middle T protein-associated tyrosine phosphokinase activity.

‣ Non-expression of a common mutation in the 21-hydroxylase gene: implications for prenatal diagnosis and carrier testing.

Rumsby, G; Massoud, A F; Avey, C; Brook, C G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1996 Português
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Mutation analysis in the family of a child with 21-hydroxylase deficiency showed that the father and affected child were homozygous for a mutation, A/C655G, believed to activate a cryptic splice site in intron 2 of the 21-hydroxylase gene. The father, who was clinically asymptomatic, showed no biochemical evidence of disease. These results create problems for the management of future pregnancies in such families and for the interpretation of the risk associated with carrier status for this mutation.

‣ Both U2 snRNA and U12 snRNA are required for accurate splicing of exon 5 of the rat calcitonin/CGRP gene

ROESSER, JAMES R.
Fonte: Copyright 2004 by RNA Society Publicador: Copyright 2004 by RNA Society
Tipo: Artigo de Revista Científica
Publicado em /08/2004 Português
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Two classes of spliceosome are present in eukaryotic cells. Most introns in nuclear pre-mRNAs are removed by a spliceosome that requires U1, U2, U4, U5, and U6 small nuclear ribonucleoprotein particles (snRNPs). A minor class of introns are removed by a spliceosome containing U11, U12, U5, U4atac, and U6 atac snRNPs. We describe experiments that demonstrate that splicing of exon 5 of the rat calcitonin/CGRP gene requires both U2 snRNA and U12 snRNA. In vitro, splicing to calcitonin/ CGRP exon 5 RNA was dependent on U2 snRNA, as preincubation of nuclear extract with an oligonucleotide complementary to U2 snRNA abolished exon 5 splicing. Addition of an oligonucleotide complementary to U12 snRNA increased splicing at a cryptic splice site in exon 5 from <5% to 50% of total spliced RNA. Point mutations in a candidate U12 branch sequence in calcitonin/CGRP intron 4, predicted to decrease U12-pre-mRNA base-pairing, also significantly increased cryptic splicing in vitro. Calcitonin/CGRP genes containing base changes disrupting the U12 branch sequence expressed significantly decreased CGRP mRNA levels when expressed in cultured cells. Coexpression of U12 snRNAs containing base changes predicted to restore U12-pre-mRNA base pairing increased CGRP mRNA synthesis to the level of the wild-type gene. These observations indicate that accurate...

‣ Determinants of the inherent strength of human 5′ splice sites

ROCA, XAVIER; SACHIDANANDAM, RAVI; KRAINER, ADRIAN R.
Fonte: Copyright 2005 by RNA Society Publicador: Copyright 2005 by RNA Society
Tipo: Artigo de Revista Científica
Publicado em /05/2005 Português
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We previously showed that the authentic 5′ splice site (5′ss) of the first exon in the human β-globin gene is intrinsically stronger than a cryptic 5′ss located 16 nucleotides upstream. Here we examined by mutational analysis the contribution of individual 5′ss nucleotides to discrimination between these two 5′ss. Based on the in vitro splicing efficiencies of a panel of 26 wild-type and mutant substrates in two separate 5′ss competition assays, we established a hierarchy of 5′ss and grouped them into three functional subclasses: strong, intermediate, and weak. Competition between two 5′ss from different subclasses always resulted in selection of the 5′ss that belongs to the stronger subclass. Moreover, each subclass has different characteristic features. Strong and intermediate 5′ss can be distinguished by their predicted free energy of base-pairing to the U1 snRNA 5′ terminus (ΔG). Whereas the extent of splicing via the strong 5′ss correlates well with the ΔG, this is not the case for competition between intermediate 5′ss. Weak 5′ss were used only when the competing authentic 5′ss was inactivated by mutation. These results indicate that extensive complementarity to U1 snRNA exerts a dominant effect for 5′ss selection...

‣ Molecular analysis of Hurler syndrome in Druze and Muslim Arab patients in Israel: multiple allelic mutations of the IDUA gene in a small geographic area.

Bach, G; Moskowitz, S M; Tieu, P T; Matynia, A; Neufeld, E F
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /08/1993 Português
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The mutations underlying Hurler syndrome (mucopolysaccharidosis IH) in Druze and Muslim Israeli Arab patients have been characterized. Four alleles were identified, using a combination of (a) PCR amplification of reverse-transcribed RNA or genomic DNA segments, (b) cycle sequencing of PCR products, and (c) restriction-enzyme analysis. One allele has two amino acid substitutions, Gly409-->Arg in exon 9 and Ter-->Cys in exon 14. The other three alleles have mutations in exon 2 (Tyr64-->Ter), exon 7 (Gln310-->Ter), or exon 8 (Thr366-->Pro). Transfection of mutagenized cDNAs into Cos-1 cells showed that two missense mutations, Thr366-->Pro and Ter-->Cys, permitted the expression of only trace amounts of alpha-L-iduronidase activity, whereas Gly409-->Arg permitted the expression of 60% as much enzyme as did the normal cDNA. The nonsense mutations were associated with abnormalities of RNA processing: (1) both a very low level of mRNA and skipping of exon 2 for Tyr64-->Ter and (2) utilization of a cryptic splice site for Gln310-->Ter. In all instances, the probands were found homozygous, and the parents heterozygous, for the mutant alleles, as anticipated from the consanguinity in each family. The two-mutation allele was identified in a family from Gaza; the other three alleles were found in seven families...

‣ Identification of cryptic splice site, exon skipping, and novel point mutations in type I CD36 deficiency

Hanawa, H; Watanabe, K; Nakamura, T; Ogawa, Y; Toba, K; Fuse, I; Kodama, M; Kato, K; Fuse, K; Aizawa, Y
Fonte: BMJ Group Publicador: BMJ Group
Tipo: Artigo de Revista Científica
Publicado em /04/2002 Português
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‣ Integrin α7β1 in Muscular Dystrophy/Myopathy of Unknown Etiology

Pegoraro, Elena; Cepollaro, Fulvio; Prandini, Paola; Marin, Alessandra; Fanin, Marina; Trevisan, Carlo P.; El-Messlemani, Abdul Hassib; Tarone, Guido; Engvall, Eva; Hoffman, Eric P.; Angelini, Corrado
Fonte: American Society for Investigative Pathology Publicador: American Society for Investigative Pathology
Tipo: Artigo de Revista Científica
Publicado em /06/2002 Português
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To investigate the role of integrin α7 in muscle pathology, we used a “candidate gene” approach in a large cohort of muscular dystrophy/myopathy patients. Antibodies against the intracellular domain of the integrin α7A and α7B were used to stain muscle biopsies from 210 patients with muscular dystrophy/myopathy of unknown etiology. Levels of α7A and α7B integrin were found to be decreased in 35 of 210 patients (∼17%). In six of these patients no integrin α7B was detected. Screening for α7B mutation in 30 of 35 patients detected only one integrin α7 missense mutation (the mutation on the second allele was not found) in a patient presenting with a congenital muscular dystrophy-like phenotype. No integrin α7 gene mutations were identified in all of the other patients showing integrin α7 deficiency. In the process of mutation analysis, we identified a novel integrin α7 isoform presenting 72-bp deletion. This isoform results from a partial deletion of exon 21 due to the use of a cryptic splice site generated by a G to A missense mutation at nucleotide position 2644 in integrin α7 cDNA. This spliced isoform is present in about 12% of the chromosomes studied. We conclude that secondary integrin α7 deficiency is rather common in muscular dystrophy/myopathy of unknown etiology...

‣ Differential splicing of human androgen receptor pre-mRNA in X-linked Reifenstein syndrome, because of a deletion involving a putative branch site.

Ris-Stalpers, C.; Verleun-Mooijman, M. C.; de Blaeij, T. J.; Degenhart, H. J.; Trapman, J.; Brinkmann, A. O.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /04/1994 Português
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The analysis of the androgen receptor (AR) gene, mRNA, and protein in a subject with X-linked Reifenstein syndrome (partial androgen insensitivity) is reported. The presence of two mature AR transcripts in genital skin fibroblasts of the patient is established, and, by reverse transcriptase-PCR and RNase transcription analysis, the wild-type transcript and a transcript in which exon 3 sequences are absent without disruption of the translational reading frame are identified. Sequencing and hybridization analysis show a deletion of > 6 kb in intron 2 of the human AR gene, starting 18 bp upstream of exon 3. The deletion includes the putative branch-point sequence (BPS) but not the acceptor splice site on the intron 2/exon 3 boundary. The deletion of the putative intron 2 BPS results in 90% inhibition of wild-type splicing. The mutant transcript encodes an AR protein lacking the second zinc finger of the DNA-binding domain. Western/immunoblotting analysis is used to show that the mutant AR protein is expressed in genital skin fibroblasts of the patient. The residual 10% wild-type transcript can be the result of the use of a cryptic BPS located 63 bp upstream of the intron 2/exon 3 boundary of the mutant AR gene. The mutated AR protein has no transcription-activating potential and does not influence the transactivating properties of the wild-type AR...

‣ Efficient splicing correction by PNA conjugation to an R6-Penetratin delivery peptide

Abes, Saïd; Turner, John J.; Ivanova, Gabriela D.; Owen, David; Williams, Donna; Arzumanov, Andrey; Clair, Philippe; Gait, Michael J.; Lebleu, Bernard
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Sequence-specific interference with the nuclear pre-mRNA splicing machinery has received increased attention as an analytical tool and for development of therapeutics. It requires sequence-specific and high affinity binding of RNaseH-incompetent DNA mimics to pre-mRNA. Peptide nucleic acids (PNA) or phosphoramidate morpholino oligonucleotides (PMO) are particularly suited as steric block oligonucleotides in this respect. However, splicing correction by PNA or PMO conjugated to cell penetrating peptides (CPP), such as Tat or Penetratin, has required high concentrations (5–10 μM) of such conjugates, unless an endosomolytic agent was added to increase escape from endocytic vesicles. We have focused on the modification of existing CPPs to search for peptides able to deliver more efficiently splice correcting PNA or PMO to the nucleus in the absence of endosomolytic agents. We describe here R6-Penetratin (in which arginine-residues were added to the N-terminus of Penetratin) as the most active of all CPPs tested so far in a splicing correction assay in which masking of a cryptic splice site allows expression of a luciferase reporter gene. Efficient and sequence-specific correction occurs at 1 μM concentration of the R6Pen–PNA705 conjugate as monitored by luciferase luminescence and by RT-PCR. Some aspects of the R6Pen–PNA705 structure–function relationship have also been evaluated.

‣ Targeted knockout and lacZ reporter expression of the mouse Tmhs deafness gene and characterization of the hscy-2J mutation

Longo-Guess, Chantal M.; Gagnon, Leona H.; Fritzsch, Bernd; Johnson, Kenneth R.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
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The Tmhs gene codes for a tetraspan transmembrane protein that is expressed in hair cell stereocilia. We previously showed that a spontaneous missense mutation of Tmhs underlies deafness and vestibular dysfunction in the hurry-scurry (hscy) mouse. Subsequently, mutations in the human TMHS gene were shown to be responsible for DFNB67, an autosomal recessive nonsyndromic deafness locus. Here we describe a genetically engineered null mutation of the mouse Tmhs gene (Tmhstm1Kjn) and show that its phenotype is identical to that of the hscy missense mutation, confirming the deleterious nature of the hscy cysteine-to-phenylalanine substitution. In the targeted null allele, the Tmhs promoter drives expression of a lacZ reporter gene. Visualization of β-galactosidase activity in Tmhstm1Kjn heterozygous mice indicates that Tmhs is highly expressed in the cochlear and vestibular hair cells of the inner ear. Expression is first detectable at E15.5, peaks around P0, decreases slightly at P6, and is absent by P15, a duration that supports the involvement of Tmhs in stereocilia development. Tmhs reporter gene expression also was detected in several cranial and cervical sensory ganglia, but not in the vestibular or spiral ganglia. We also describe a new nontargeted mutation of the Tmhs gene...

‣ Tnni3k Modifies Disease Progression in Murine Models of Cardiomyopathy

Wheeler, Ferrin C.; Tang, Hao; Marks, Odessa A.; Hadnott, Tracy N.; Chu, Pei-Lun; Mao, Lan; Rockman, Howard A.; Marchuk, Douglas A.
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
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The Calsequestrin (Csq) transgenic mouse model of cardiomyopathy exhibits wide variation in phenotypic progression dependent on genetic background. Seven heart failure modifier (Hrtfm) loci modify disease progression and outcome. Here we report Tnni3k (cardiac Troponin I-interacting kinase) as the gene underlying Hrtfm2. Strains with the more susceptible phenotype exhibit high transcript levels while less susceptible strains show dramatically reduced transcript levels. This decrease is caused by an intronic SNP in low-transcript strains that activates a cryptic splice site leading to a frameshifted transcript, followed by nonsense-mediated decay of message and an absence of detectable protein. A transgenic animal overexpressing human TNNI3K alone exhibits no cardiac phenotype. However, TNNI3K/Csq double transgenics display severely impaired systolic function and reduced survival, indicating that TNNI3K expression modifies disease progression. TNNI3K expression also accelerates disease progression in a pressure-overload model of heart failure. These combined data demonstrate that Tnni3k plays a critical role in the modulation of different forms of heart disease, and this protein may provide a novel target for therapeutic intervention.

‣ Investigation of age-related changes in LMNA splicing and expression of progerin in human skeletal muscles

Luo, Yue-Bei; Mitrpant, Chalermchai; Johnsen, Russell D; Fabian, Victoria A; Fletcher, Sue; Mastaglia, Frank L; Wilton, Steve D
Fonte: e-Century Publishing Corporation Publicador: e-Century Publishing Corporation
Tipo: Artigo de Revista Científica
Publicado em 15/11/2013 Português
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Age-related changes in splice-forms of LMNA, which encodes the nuclear lamina proteins lamin A/C, have not been investigated in skeletal muscle. In the rare premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS), de novo point mutations in LMNA activate a cryptic splice site in exon 11, resulting in a 150 base deletion in LMNA mRNA and accumulation of a truncated protein isoform, progerin. The LMNA Δ150 progerin transcript has also been found in trace quantities in tissues of healthy people and its implication in ‘natural’ ageing has been proposed. We therefore investigated the expression of progerin and lamin A/C in normal human and mouse skeletal muscles of different ages. LMNA Δ150 was detected in most muscle samples from healthy individuals aged 16-71 years, but was not present in any mouse muscle samples up to the age of 18 months. Real time qPCR of human muscle samples showed that there was an age-related increase in both the full length lamin A and LMNA Δ150 transcripts, whereas their protein levels did not change significantly with age. These findings indicate that there is a basal level of mis-splicing during LMNA expression that does not change with ageing in human muscle, but at levels that do not result in increased aberrant protein. The significance of these findings in the pathophysiology of muscle ageing is uncertain and warrants further investigation.

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

Winnard, A V; Mendell, J R; Prior, T W; Florence, J; Burghes, A H
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /01/1995 Português
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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.

‣ Deficiency of FRAS1-related extracellular matrix 1 (FREM1) causes congenital diaphragmatic hernia in humans and mice

Beck, Tyler F.; Veenma, Danielle; Shchelochkov, Oleg A.; Yu, Zhiyin; Kim, Bum Jun; Zaveri, Hitisha P.; van Bever, Yolande; Choi, Sunju; Douben, Hannie; Bertin, Terry K.; Patel, Pragna I.; Lee, Brendan; Tibboel, Dick; de Klein, Annelies; Stockton, David W.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
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Congenital diaphragmatic hernia (CDH) is a common life-threatening birth defect. Recessive mutations in the FRAS1-related extracellular matrix 1 (FREM1) gene have been shown to cause bifid nose with or without anorectal and renal anomalies (BNAR) syndrome and Manitoba oculotrichoanal (MOTA) syndrome, but have not been previously implicated in the development of CDH. We have identified a female child with an isolated left-sided posterolateral CDH covered by a membranous sac who had no features suggestive of BNAR or MOTA syndromes. This child carries a maternally-inherited ∼86 kb FREM1 deletion that affects the expression of FREM1's full-length transcripts and a paternally-inherited splice site mutation that causes activation of a cryptic splice site, leading to a shift in the reading frame and premature termination of all forms of the FREM1 protein. This suggests that recessive FREM1 mutations can cause isolated CDH in humans. Further evidence for the role of FREM1 in the development of CDH comes from an N-ethyl-N-nitrosourea -derived mouse strain, eyes2, which has a homozygous truncating mutation in Frem1. Frem1eyes2 mice have eye defects, renal agenesis and develop retrosternal diaphragmatic hernias which are covered by a membranous sac. We confirmed that Frem1 is expressed in the anterior portion of the developing diaphragm and found that Frem1eyes2 embryos had decreased levels of cell proliferation in their developing diaphragms when compared to wild-type embryos. We conclude that FREM1 plays a critical role in the development of the diaphragm and that FREM1 deficiency can cause CDH in both humans and mice.

‣ Mutations in the lysosomal beta-galactosidase gene that cause the adult form of GM1 gangliosidosis.

Chakraborty, S.; Rafi, M. A.; Wenger, D. A.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1994 Português
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Three adult patients with acid beta-galactosidase deficiency/GM1 gangliosidosis who were from two unrelated families of Scandinavian descent were found to share a common point mutation in the coding region of the corresponding gene. The patients share common clinical features, including early dysarthria, mild ataxia, and bone abnormalities. When cDNA from the two patients in family 1 was PCR amplified and sequenced, most (39/41) of the clones showed a C-to-T transition (C-->T) at nucleotide 245 (counting from the initiation codon). This mutation changes the codon for Thr(ACG) to Met(ATG). Mutant and normal sequences were also found in that position in genomic DNA, indicating the presence of another mutant allele. Genomic DNA from the patient in family 2 revealed the same point mutation in one allele. It was determined that in each family only the father carried the C-->T mutation. Expression studies showed that this mutation produced 3%-4% of beta-galactosidase activity, confirming its deleterious effects. The cDNA clones from the patients in family 1 that did not contain the C-->T revealed a 20-bp insertion of intronic sequence between nucleotides 75 and 76, the location of the first intron. Further analysis showed the insertion of a T near the 5' splice donor site which led to the use of a cryptic splice site. It appears that the C-->T mutation results in enough functional enzyme to produce a mild adult form of the disease...

‣ Seven novel mutations in the methylenetetrahydrofolate reductase gene and genotype/phenotype correlations in severe methylenetetrahydrofolate reductase deficiency.

Goyette, P; Frosst, P; Rosenblatt, D S; Rozen, R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1995 Português
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5-Methyltetrahydrofolate, the major form of folate in plasma, is a carbon donor for the remethylation of homocysteine to methionine. This form of folate is generated from 5,10-methylenetetrahydrofolate through the action of 5,10-methylenetetrahydrofolate reductase (MTHFR), a cytosolic flavoprotein. Patients with an autosomal recessive severe deficiency of MTHFR have homocystinuria and a wide range of neurological and vascular disturbances. We have recently described the isolation of a cDNA for MTHFR and the identification of two mutations in patients with severe MTHFR deficiency. We report here the characterization of seven novel mutations in this gene: six missense mutations and a 5' splice-site defect that activates a cryptic splice site in the coding sequence. We also present a preliminary analysis of the relationship between genotype and phenotype for all nine mutations identified thus far in this gene. A nonsense mutation and two missense mutations (proline to leucine and threonine to methionine) in the homozygous state are associated with extremely low activity (0%-3%) and onset of symptoms within the 1st year of age. Other missense mutations (arginine to cysteine and arginine to glutamine) are associated with higher enzyme activity and later onset of symptoms.