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Splicing Pattern (splicing + pattern)
Selected AbstractsEx vivo splicing assays of mutations at noncanonical positions of splice sites in USHER genes,HUMAN MUTATION, Issue 3 2010Sandie Le Guédard-Méreuze Abstract Molecular diagnosis in Usher syndrome type 1 and 2 patients led to the identification of 21 sequence variations located in noncanonical positions of splice sites in MYO7A, CDH23, USH1C, and USH2A genes. To establish experimentally the splicing pattern of these substitutions, whose impact on splicing is not always predictable by available softwares, ex vivo splicing assays were performed. The branch-point mapping strategy was also used to investigate further a putative branch-point mutation in USH2A intron 43. Aberrant splicing was demonstrated for 16 of the 21 (76.2%) tested sequence variations. The mutations resulted more frequently in activation of a nearby cryptic splice site or use of a de novo splice site than exon skipping (37.5%). This study allowed the reclassification as splicing mutations of one silent (c.7872G>A (p.Glu2624Glu) in CDH23) and four missense mutations (c.2993G>A (p.Arg998Lys) in USH2A, c.592G>A (p.Ala198Thr), c.3503G>C [p.Arg1168Pro], c.5944G>A (p.Gly1982Arg) in MYO7A), whereas it provided clues about a role in structure/function in four other cases: c.802G>A (p.Gly268Arg), c.653T>A (p.Val218Glu) (USH2A), and c.397C>T (p.His133Tyr), c.3502C>T (p.Arg1168Trp) (MYO7A). Our data provide insights into the contribution of splicing mutations in Usher genes and illustrate the need to define accurately their splicing outcome for diagnostic purposes. Hum Mutat 31:1,9, 2010. © 2010 Wiley-Liss, Inc. [source] A human-specific mutation leads to the origin of a novel splice form of neuropsin (KLK8), a gene involved in learning and memory,HUMAN MUTATION, Issue 10 2007Zhi-xiang Lu Abstract Neuropsin (kallikrein 8, KLK8) is a secreted-type serine protease preferentially expressed in the central nervous system and involved in learning and memory. Its splicing pattern is different in human and mouse, with the longer form (type II) only expressed in human. Sequence analysis suggested a recent origin of type II during primate evolution. Here we demonstrate that the type II form is absent in nonhuman primates, and is thus a human-specific splice form. With the use of an in vitro splicing assay, we show that a human-specific T to A mutation (c.71,127T>A) triggers the change of splicing pattern, leading to the origin of a novel splice form in the human brain. Using mutation assay, we prove that this mutation is not only necessary but also sufficient for type II expression. Our results demonstrate a molecular mechanism for the creation of novel proteins through alternative splicing in the central nervous system during human evolution. Hum Mutat 28(10), 978,984, 2007. © 2007 Wiley-Liss, Inc. [source] Phenotypic consequences of branch point substitutions,HUMAN MUTATION, Issue 8 2006Jana Královi Abstract The branch point sequence (BPS) is a conserved splicing signal important for spliceosome assembly and lariat intron formation. BPS mutations may result in aberrant pre-mRNA splicing and genetic disorders, but their phenotypic consequences have been difficult to predict, largely due to a highly degenerate nature of the BPS consensus. Here, we have examined the splicing pattern of nine reporter pre-mRNAs that have previously been shown to give rise to human hereditary diseases as a result of single-nucleotide substitutions in the predicted BPS. Increased exon skipping and intron retention observed in vivo were recapitulated for each mutated pre-mRNA, but the reproducibility of cryptic splice site activation was lower. BP mutations in reporter pre-mRNAs frequently induced aberrant 3, splice sites and also activated a cryptic 5, splice site. Systematic mutagenesis of BP adenosines showed that in most pre-mRNAs, the expression of canonical transcripts was lower for BP transitions than BP transversions. Differential splicing outcome for transitions vs. transversions was abrogated or reduced if introns were truncated to 200 nt or less, suggesting that the nature of the BP residue is less critical for interactions across very short introns. Together, these results improve prediction of phenotypic consequences of point mutations upstream of splice acceptor sites and suggest that the overrepresentation of disease-causing adenosine-to-guanosine BP substitutions observed in Mendelian disorders is due to more profound defects of gene expression at the level of pre-mRNA splicing. Hum Mutat 27(8), 803,813, 2006. © 2006 WileyLiss, Inc. [source] Band 4.1 proteins are expressed in the retina and interact with both isoforms of the metabotropic glutamate receptor type 8JOURNAL OF NEUROCHEMISTRY, Issue 6 2008Melanie Rose Abstract The function of the CNS depends on the correct regulation of neurotransmitter receptors by interacting proteins. Here, we screened a retinal cDNA library for proteins interacting with the intracellular C-terminus of the metabotropic glutamate receptor isoform 8a (mGluR8a). The band 4.1B protein binds to the C-termini of mGluR8a and mGluR8b, co-localizes with these glutamate receptors in transfected mammalian cells, facilitates their cell surface expression and inhibits the mGluR8 mediated reduction of intracellular cAMP concentrations. In contrast, no interaction with 4.1B was observed for other mGluRs tested. Amino acids encoded by exons 19 and 20 of 4.1B and a stretch of four basic amino acids present in the mGluR8 C-termini mediate the protein interaction. Besides binding to 4.1B, mGluR8 isoforms interact with 4.1G, 4.1N, and 4.1R. Because band 4.1 transcripts undergo extensive alternative splicing, we analyzed the splicing pattern of interacting regions and detected a 4.1B isoform expressed specifically in the retina. Within this tissue, mGluR8 and 4.1B, 4.1G, 4.1N, and 4.1R show a comparable distribution, being expressed in both synaptic layers and in somata of the ganglion cell layer. In summary, our studies identified band 4.1 proteins as new players for the mGluR8 mediated signal transduction. [source] Using a minigene approach to characterize a novel splice site mutation in human F7 gene causing inherited factor VII deficiency in a Chinese pedigreeHAEMOPHILIA, Issue 6 2009T. YU Summary., Factor VII deficiency which transmitted as an autosomal recessive disorder is a rare haemorrhagic condition. The aim of this study was to identify the molecular genetic defect and determine its functional consequences in a Chinese pedigree with FVII deficiency. The proband was diagnosed as inherited coagulation FVII deficiency by reduced plasma levels of FVII activity (4.4%) and antigen (38.5%). All nine exons and their flanking sequence of F7 gene were amplified by polymerase chain reaction (PCR) for the proband and the PCR products were directly sequenced. The compound heterozygous mutations of F7 (NM_000131.3) c.572-1G>A and F7 (NM_000131.3) c.1165T>G; p.Cys389Gly were identified in the proband's F7 gene. To investigate the splicing patterns associated with F7 c.572-1G>A, ectopic transcripts in leucocytes of the proband were analyzed. F7 minigenes, spanning from intron 4 to intron 7 and carrying either an A or a G at position -1 of intron 5, were constructed and transiently transfected into human embryonic kidney (HEK) 293T cells, followed by RT-PCR analysis. The aberrant transcripts from the F7 c.572-1G>A mutant allele were not detected by ectopic transcription study. Sequencing of the RT-PCR products from the mutant transfectant demonstrated the production of an erroneously spliced mRNA with exon 6 skipping, whereas a normal splicing occurred in the wide type transfectant. The aberrant mRNA produced from the F7 c.572-1G>A mutant allele is responsible for the factor VII deficiency in this pedigree. [source] The role of the RNA-binding protein Sam68 in mammary tumourigenesis,THE JOURNAL OF PATHOLOGY, Issue 3 2010David J Elliott Abstract The RNA binding protein Sam68 (Src- associated in mitosis 68 kD) is implicated in cell signalling, transcriptional regulation, pre-mRNA splicing, and is overexpressed and/or hyperphosphorylated in breast, prostate, and renal cancers. Sam68 has roles in normal breast development; however, a study by Song et al published in this issue of The Journal of Pathology reports overexpression of nuclear and cytoplasmic Sam68 protein in a large cohort of clinical breast tumours, implicating Sam68 as a potential prognostic indicator and target for therapy. In breast cancer cells, nuclear Sam68 protein might affect the expression of cancer-relevant genes and/or modulate exon splicing patterns in a dose-dependent manner. Sam68-regulated expression of alternative transcripts may help drive mammary tumourigenesis. The high levels of cytoplasmic Sam68 protein observed in breast cancer cells could also impact on cellular signalling pathways important for mammary tumour cell biology. Copyright © 2010 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Invited Commentary for Song L et al. Sam68 up-regulation correlates with, and its down-regulation inhibits, proliferation and tumourigenicity of breast cancer cells. Journal of Pathology, http://dx.doi.org/10.1002/path.2751 [source] Non-SCN5A Related Brugada Syndromes: Verification of Normal Splicing and Trafficking of SCN5A Without Exonic MutationsANNALS OF HUMAN GENETICS, Issue 1 2007Yukiko Nakano Summary Recently, it has been reported that under 20% of Brugada syndrome cases are linked to SCN5A mutations. The purpose of this study was to clarify whether abnormalities other than exonic mutations, such as splicing disorders, decreased mRNA expression levels, or membrane transport abnormalities of SCN5A, play a role in the pathogenesis of Brugada syndrome. We analyzed all SCN5A exons and splice sites using genomic DNA from 23 Brugada syndrome patients. We also analyzed the mRNA obtained from RV cardiomyocytes using real time PCR and sequencing, to study the expression levels and splicing patterns of SCN5A. The localization of SCN5A was examined by immunofluorescence analysis. A de novo heterozygous G to A transversion in a 5, splice junction of the intron between exons 21 and 22 was detected in 1 patient. In the mRNA analysis of Brugada syndrome patients without a mutation of SCN5A no splicing abnormalities were detected, and the SCN5A mRNA levels were similar to those of normal controls. Immunofluorescence analyses revealed that SCN5A is located on the surface membrane not only in the RV cardiomyocytes of normal controls but also in those with Brugada syndrome. We can confirm that some Brugada syndrome patients without exonic mutations in SCN5A had no other SCN5A abnormalities, including any involving the location of the SCN5A protein. These results suggest the involvement of other proteins in the pathogenesis in Brugada syndrome. [source] Mutational screening of the CYP26A1 gene in patients with caudal regression syndrome,BIRTH DEFECTS RESEARCH, Issue 2 2006Patrizia De Marco Abstract BACKGROUND The retinoic acid (RA),catabolizing enzyme Cyp26a1 plays an important role in protecting tailbud tissues from inappropriate exposure to RA. Cyp26a1 -null animals exhibit caudal agenesis and spina bifida, imperforate anus, agenesis of the caudal portions of the digestive and urogenital tracts, and malformed lumbosacral skeletal elements. This phenotype closely resembles the most severe form of caudal agenesis in humans. In view of these findings, we investigated a potential involvement of the human CYP26A1 gene in the pathogenesis of caudal regression syndrome (CRS). METHODS Mutational screening of 49 CRS patients and 132 controls was performed using denaturing high-performance liquid chromatography and sequencing. Differences in the genotype and allele frequency of each SNP were evaluated by ,2 analysis. The biological significance of the intronic variants was investigated by transfection assays of mutant constructs and by analysis of the splicing patterns with RT-PCR. RESULTS Mutational screening allowed us to identify 6 SNPs, 4 of which (447C>G, 1134G>A, IVS1+10G>C, and IVS4+8AG>GA) are new. In addition, we describe a novel 2-site haplotype consisting of the 2 intronic SNPs. Both single-locus and haplotype analyses revealed no association with increased risk for CRS. The consequences of the 2 intronic polymorphisms on the mRNA splicing process were also investigated. Moreover, using functional and computational methods we demonstrated that both of these intronic polymorphisms affect the intron splicing efficiency. CONCLUSIONS Our research did not provide evidence that CYP26A1 has implications for the pathogenesis of human CRS. However, the relationship between CRS risk and the CYP26A1 genotype requires further study with a larger number of genotyped subjects. Birth Defects Research (Part A), 2006. © 2006 Wiley-Liss, Inc. [source] | |||||||||||||