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Results for “Splicing mutations”

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PubMed1984

Normal and mutant human beta-globin pre-mRNAs are faithfully and efficiently spliced in vitro.

Human beta-globin mRNA precursors (pre-mRNAs) synthesized in vitro from a bacteriophage SP6 promoter/beta-globin gene fusion are accurately and efficiently spliced when added to a HeLa cell nuclear extract. Under optimal conditions, the first intervening sequence (IVS 1) is removed by splicing in up to 90% of the input pre-mRNA. Splicing requires ATP and in its absence the pre-mRNA is neither spliced nor cleaved at splice junctions. Splicing does not require that the pre-mRNA contain a correct 5' or 3' end, a 3' poly A tail, or a 5'-terminal cap structure. However, capping of the pre-mRNA significantly affects the specificity of in vitro processing. In the absence of a cap approximately 30%-40% of the pre-mRNA is accurately spliced, and a number of aberrantly cleaved RNAs are also detected. In contrast, capped pre-mRNAs are spliced more efficiently and produce fewer aberrant RNA species. The specificity of splice-site selection in vitro was tested by analyzing pre-mRNAs that contain beta-thalassemia splicing mutations in IVS 1. Remarkably, these mutations cause the same abnormal splicing events in vitro and in vivo. The ability to synthesize mutant pre-mRNAs and study their splicing in a faithful in vitro system provides a powerful approach to determine the mechanisms of RNA splice-site selection.

Adenosine Triphosphate↗

PubMed1994

An exon 8-spliced out transcript of neurofibromatosis 2 gene is constitutively expressed in various human tissues.

We succeeded in cloning the exon 8-spliced out transcript of neurofibromatosis 2 (NF2) gene in human glioma cell lines. We then investigated the expression of the spliced out form in various human tissues by PCR analysis followed by Southern blot hybridization and sequencing to evaluate whether it was generated by normal alternative splicing or by a splicing mutation. The analysis revealed the splicing out of exon 8 in the various tissues, and the transcript missing exon 8 should thus be considered as a product of normal alternative splicing. Our results further support the possibility that the NF2 gene expresses multiple alternatively-spliced transcripts variantly in different tissue types.

Base Sequence↗

PubMed2000

Three novel types of splicing aberrations in the tuberous sclerosis TSC2 gene caused by mutations apart from splice consensus sequences.

Disease causing aberrations in both tuberous sclerosis predisposing genes, TSC1 and TSC2, comprise nearly every type of alteration with a predominance of small truncating mutations distributed over both genes. We performed an RNA based screening of the entire coding regions of both TSC genes applying the protein truncation test (PTT) and identified a high proportion of unusual splicing abnormalities affecting the TSC2 gene. Two cases exhibited different splice acceptor mutations in intron 9 (IVS9-15G-->A and IVS9-3C-->G) both accompanied by exon 10 skipping and simultaneous usage of a cryptic splice acceptor in exon 10. Another splice acceptor mutation (IVS38-18A-->G) destroyed the putative polypyrimidine structure in intron 38 and resulted in simultaneous intron retention and usage of a downstream cryptic splice acceptor in exon 39. Another patient bore a C-->T transition in intron 8 (IVS8+281C-->T) activating a splice donor site and resulting in the inclusion of a newly recognised exon in the mRNA followed by a premature stop. These splice variants deduced from experimental results are additionally supported by RNA secondary structure analysis based on free energy minimisation. Three of the reported splicing anomalies are due to sequence changes remote from exon/intron boundaries, described for the first time in TSC. These findings highlight the significance of investigating intronic changes and their consequences on the mRNA level as disease causing mutations in TSC.

Base Sequence↗

PubMed1993

Hprt activities and RNA phenotypes in 6-thioguanine resistant human T-lymphocytes.

The phenotypic effects of mutation in the hypoxanthine phosphoribosyltransferase (hprt) gene on hprt enzyme activity and hprt mRNA levels were studied in 6-thioguanine (TG) resistant human T-cell clones with various types of hprt mutation. The mean enzyme activity in 16 TG selected clones was less than 1% of the mean in unselected clones. The hprt mRNA levels, measured by a quantitative RNA/RNA solution hybridization assay, were within the normal range in 38% of the mutant clones. Reduced hprt mRNA levels were found in all of three nonsense mutations, four out of five splicing mutations, both of two clones with genomic alterations, three out of five missense mutations and one out of four frameshift mutations caused by 1-4-bp deletions. Intermediate and high enzyme activity and normal hprt mRNA levels were found in two TG selected clones where no hprt mutation was detected. Several clones with very low hprt mRNA levels were found to yield hprt cDNA by PCR amplification. These results show that hprt mutation leads to decreased steady state levels of hprt mRNA in a majority of TG resistant T-cell clones, and that many different types of hprt mutation can have this effect.

Drug Resistance↗

PubMed2006

Novel spastin (SPG4) mutations in Italian patients with hereditary spastic paraplegia.

Spastic paraplegia type 4 is caused by mutations in the gene that encodes spastin (SPG4), a member of the AAA protein family. A cohort of 34 unrelated Italian patients with pure spastic paraplegia, of which 18 displayed autosomal dominant inheritance and 16 were apparently sporadic, were screened for mutations in the SPG4 gene by denaturing high performance liquid chromatography. We identified a previously reported mutation in a sporadic patient with pure hereditary spastic paraplegia. We also identified eight unrelated patients with pure autosomal dominant hereditary spastic paraplegia carrying five novel mutations in the SPG4 gene (one missense mutation, c.1304 C>T; one nonsense mutation, c.807C>A; two frameshift mutations, c.1281dupT, c.1514_1515insATA; and one splicing mutation, c.1322-2A>C). The frequency for SPG4 mutations detected in autosomal dominant hereditary spastic paraplegia was 44.4%. This study contributes to expand the spectrum of SPG4 mutations in Italian population.

Adenosine Triphosphatases↗

PubMed2003

Collagen XI sequence variations in nonsyndromic cleft palate, Robin sequence and micrognathia.

Cleft palate is a common birth defect, but its etiopathogenesis is mostly unknown. Several studies have shown that cleft palate has a strong genetic component. Robin sequence consists of three of the following four findings: micrognathia, glossoptosis, obstructive apnea, and cleft palate. While cleft palate is mainly nonsyndromic, about 80 percent of Robin sequence cases are associated with syndromes. Mutations in genes coding for cartilage collagens II and XI, COL2A1, COL11A1 and COL11A2, have been shown to cause chondrodysplasias that are commonly associated with Robin sequence, micrognathia or cleft palate. We therefore analyzed a cohort of 24 patients with nonsyndromic Robin sequence, 17 with nonsyndromic cleft palate and 21 with nonsyndromic micrognathia for mutations in COL11A2. A total of 23 Robin sequence patients were also analyzed for mutations in COL2A1 and COL11A1. We detected two disease-associated mutations in patients with Robin sequence, an Arg to stop codon mutation in COL11A2 and a splicing mutation in COL11A1. Two putatively disease-associated sequence variations were found in COL11A1 in Robin sequence patients, one in COL11A2 in a patient with micrognathia and one in COL2A1 in two patients with Robin sequence. The results showed that sequence variations in these genes can play a role in the etiology of Robin sequence, cleft palate and micrognathia but are not common causes of these phenotypes.

Cleft Palate↗

PubMed2000

Structure of the SLC7A7 gene and mutational analysis of patients affected by lysinuric protein intolerance.

Lysinuric protein intolerance (LPI) is a rare autosomal recessive defect of cationic amino acid transport caused by mutations in the SLC7A7 gene. We report the genomic structure of the gene and the results of the mutational analysis in Italian, Tunisian, and Japanese patients. The SLC7A7 gene consists of 10 exons; sequences of all of the exon-intron boundaries are reported here. All of the mutant alleles were characterized and eight novel mutations were detected, including two missense mutations, 242A-->C (M1L) and 1399C-->A (S386R); a nonsense mutation 967G-->A (W242X); two splice mutations IVS3 +1G-->A and IVS6 +1G-->T; a single-base insertion, 786insT; and two 4-bp deletions, 455delCTCT and 1425delTTCT. In addition, a previously reported mutation, 1625insATCA, was found in one patient. It is noteworthy that 242A-->C causes the change of Met1 to Leu, a rare mutational event previously found in a few inherited conditions. We failed to establish a genotype/phenotype correlation. In fact, both intrafamilial and interfamilial phenotypic variability were observed in homozygotes for the same mutation. The DNA-based tests are now easily accessible for molecular diagnosis, genetic counseling, and prenatal diagnosis of LPI.

Amino Acid Metabolism, Inborn Errors↗

PubMed1997

Mutation screening of the EXT1 and EXT2 genes in patients with hereditary multiple exostoses.

Hereditary multiple exostoses (HME), the most frequent of all skeletal dysplasias, is an autosomal dominant disorder characterized by the presence of multiple exostoses localized mainly at the end of long bones. HME is genetically heterogeneous, with at least three loci, on 8q24.1 (EXT1), 11p11-p13 (EXT2), and 19p (EXT3). Both the EXT1 and EXT2 genes have been cloned recently and define a new family of potential tumor suppressor genes. This is the first study in which mutation screening has been performed for both the EXT1 and EXT2 genes prior to any linkage analysis. We have screened 17 probands with the HME phenotype, for alterations in all translated exons and flanking intronic sequences, in the EXT1 and EXT2 genes, by conformation-sensitive gel electrophoresis. We found the disease-causing mutation in 12 families (70%), 7 (41%) of which have EXT1 mutations and 5 (29%) EXT2 mutations. Together with the previously described 1-bp deletion in exon 6, which is present in 2 of our families, we report five new mutations in EXT1. Two are missense mutations in exon 2 (G339D and R340C), and the other three alterations (a nonsense mutation, a frameshift, and a splicing mutation) are likely to result in truncated nonfunctional proteins. Four new mutations are described in EXT2. A missense mutation (D227N) was found in 2 different families; the other three alterations (two nonsense mutations and one frameshift mutation) lead directly or indirectly to premature stop codons. The missense mutations in EXT1 and EXT2 may pinpoint crucial domains in both proteins and therefore give clues for the understanding of the pathophysiology of this skeletal disorder.

Electrophoresis, Polyacrylamide Gel↗

PubMed1991

Mutations in collagen genes: causes of rare and some common diseases in humans.

More than 70 mutations in the two structural genes for type I procollagen (COL1A1 and COL1A2) have been found in probands with osteogenesis imperfecta, a heritable disease of children characterized by fragility of bone and other tissues rich in type I collagen. The mutations include deletions, insertions, RNA splicing mutations, and single-base substitutions that convert a codon for glycine to a codon for an amino acid with a bulkier side chain. With a few exceptions, the most severe phenotypes of the disease are explained largely by synthesis of structurally defective pro alpha chains of type I procollagen that either interfere with the folding of the triple helix or with self-assembly of collagen into fibrils. The results emphasize the extent to which the zipperlike folding of the collagen triple helix and the self-assembly of collagen fibrils depend on the principle of nucleated growth whereby a few subunits form a nucleus and the nucleus is then propagated to generate a large structure with a precisely defined architecture. The principle of nucleated growth is a highly efficient mechanism for the assembly of large structures, but biological systems that depend extensively on nucleated growth are highly vulnerable to mutations that cause synthesis of structurally abnormal but partially functional subunits. Recently, several mutations in three other collagen genes (COL2A1, COL3A1, and COL4A5) have been found in probands with genetic diseases involving tissues rich in these collagens. Most of the probands have rare genetic diseases but a few appear to have phenotypes that are difficult to distinguish from more common disorders such as osteoarthritis, osteoporosis, and aortic aneurysms. Therefore, the results suggest that mutations in procollagen genes may cause a wide spectrum of both rare and common human diseases.

Animals↗

PubMedDate not supplied

Study of 4-NQO induced mutations at the HPRT locus in CHO cell lines. Possible influences of preferential DNA-repair on the mutational spectrum.

In the attempt to determine the possible influence of excision repair processes on 4-NQO mutational spectra in mammalian cells, 4-NQO-induced mutants at the hprt locus were isolated in excision repair proficient (AA8) and deficient (UV5) CHO cell lines. DNA sequencing data on these mutants revealed that DNA repair may indeed modulate the induced mutational spectrum. In particular, more splice mutations were found in the repair proficient than in the repair deficient cells. This can be interpreted by a difference in repairability of the two principal 4-NQO G-adducts or by the existence of a transcription-coupled DNA preferential repair process.

4-Nitroquinoline-1-oxide↗

PubMed1997

[alpha-Galactosidase gene mutation and its expression product in Fabry disease (alpha-galactosidase deficiency)].

Fabry disease is characterized by a deficiency of lysosomal alpha-galactosidase (alpha-Gal) and the accumulation of glycosphingolipid (e.g. predominantly globotriaosylceramide) in various tissues, mainly in lysosomes of the vascular endothelium. This disorder is currently classified into two clinical phenotypes; classical severe type and atypical variant type. Classical form patients, with clinical manifestations of generalized angiopathy of early onset, usually show no detectable alpha-Gal activity. Recently, there are also atypical form patients with residual alpha-Gal activity and late-onset cardiomyopathy without other systemic manifestations. So far, we identified a number of alpha-Gal gene mutations including partial gene deletions, splicing mutations, nonsense mutations and missense mutations. They were heterogeneous and more than half of them were missense mutations. To clarify the molecular mechanism causing the enzyme defect in the patient, various missense mutations were expressed in COS-1 cells. At least, two groups have been identified; one expressing a mutant enzyme without catalytic activity (non-functional type), and the other expressing catalytically active but unstable mutant enzyme (fragile type). The fragile type mutants were widely present in the different clinical phenotypes from classical severe type to atypical milder type including subclinical Fabry hemizygote, and the mutant enzymes were posttranslationally inactivated and degraded in the cells. The inactivation and degradation were prevented by the addition of substrate analogue; galactose or melibiose. These findings provided us with significant informations on the molecular pathology of the enzyme defect in Fabry disease, and suggested the possibility of a new therapeutic approach for this disease.

Fabry Disease↗

PubMed1998

[Molecular analysis of peroxisomal disorders].

Peroxisome biogenesis disorders (PBD) include Zellweger syndrome (ZS), neonatal adrenoleukodystrophy (NALD) and infantile Refsum disease (IRD). They are classified into ten complementation groups. Five pathogenic genes have been identified using different model systems of peroxisome deficient mutants. PAF-1 and 2 were identified from CHO mutants and were responsible genes for PBD group F and C. Human PEX 5, 12 and 1, responsible genes for group 2, 3 and 1, respectively, were cloned by homology search between yeast PEX genes and human genes on the cDNA data base. Adrenoleukodystrophy (ALD), the most frequent peroxisomal disorder, shows phenotypic heterogeneity. Its responsible gene was cloned by positional cloning. It encodes a 75 kDa peroxisomal membrane protein (ALDP) that is a member of the ATP-binding cassette transporter family. There are about 120 different mutations including missense, nonsense and splice mutations, as well as insertions and deletions of a few base pairs. There is no correlation between the clinical phenotype and the ALDP gene mutation. Recently, animal models have been produced by targeted mutation of the PBD and ALD genes. The mouse model should facilitate researches on PBD and ALD, especially those on regulatory factors of their phenotypic heterogeneity and on new therapeutic approaches.

Adrenoleukodystrophy↗

PubMed2006

RBM4 interacts with an intronic element and stimulates tau exon 10 inclusion.

Tau protein, which binds to and stabilizes microtubules, is critical for neuronal survival and function. In the human brain, tau pre-mRNA splicing is regulated to maintain a delicate balance of exon 10-containing and exon 10-skipping isoforms. Splicing mutations affecting tau exon 10 alternative splicing lead to tauopathies, a group of neurodegenerative disorders including dementia. Molecular mechanisms regulating tau alternative splicing remain to be elucidated. In this study, we have developed an expression cloning strategy to identify splicing factors that stimulate tau exon 10 inclusion. Using this expression cloning approach, we have identified a previously unknown tau exon 10 splicing regulator, RBM4 (RNA binding motif protein 4). In cells transfected with a tau minigene, RBM4 overexpression leads to an increased inclusion of exon 10, whereas RBM4 down-regulation decreases exon 10 inclusion. The activity of RBM4 in stimulating tau exon 10 inclusion is abolished by mutations in its RNA-binding domain. A putative intronic splicing enhancer located in intron 10 of the tau gene is required for the splicing stimulatory activity of RBM4. Immunohistological analyses reveal that RBM4 is expressed in the human brain regions affected in tauopathy, including the hippocampus and frontal cortex. Our study demonstrates that RBM4 is involved in tau exon 10 alternative splicing. Our work also suggests that down-regulating tau exon 10 splicing activators, such as RBM4, may be of therapeutic potential in tauopathies involving excessive tau exon 10 inclusion.

Adult↗

PubMed1996

RNA-based mutation screening in hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is a cancer syndrome inherited in an autosomal dominant fashion. Four susceptibility genes are known, which code for DNA mismatch repair enzymes. The purpose of this study was to identify the HNPCC gene defects in a cohort of Australian HNPCC families and to evaluate the use of RNA-based screening methods. Six mutations were identified, four in the hMLH1 gene and two in hMSH2, by using a combination of DNA-based and RNA-based methods. One of the hMLH1 defects was a missense mutation, and the other five mutations would be expected to result in a shortened protein. These included a rare type of mRNA splicing mutation in hMLH1 exon 17. By use of reverse-transcriptase (RT) PCR, defective transcripts were detectable for three of the hMLH1 mutations but not for the fourth one, which was predicted to cause skipping of exon 15. Furthermore, many more alternative transcripts for the hMLH1 gene were found than previously described, and these were more abundant in the RNA samples prepared from whole blood than from lymphoblastoid cell lines. This confounded RNA-based screening for HNPCC mutations, because it was difficult to determine which aberrant RT-PCR fragment was the real hereditary defect. One of the splice-site mutations reported here causes skipping of exons 9 and 10, which also occurs as an alternative transcript. When the protein-truncation test was used, the results were indistinguishable between the patients in this family and controls. Other aberrant transcripts were also observed that varied in size between individuals but were unrelated to the hereditary defects. This study has important implications for the design of reliable diagnostic tests for HNPCC gene defects.

Cell Line↗

PubMed2001

Analysis of splicing parameters in the dystrophin gene: relevance for physiological and pathogenetic splicing mechanisms.

The molecular mechanisms that direct splice-site selection and assure orderly exon juxtaposition have not been fully clarified. The extraordinary nature of the dystrophin gene points to several hurdles in the processing of transcripts. In this study, dystrophin statistical and thermodynamic splicing parameters have been evaluated providing the first comprehensive description for a single human gene. We show that concomitant use of consensus values (CV) and DeltaDG degrees 37 values for U1snRNA annealing better discriminates between real donor sites and donor-like sequences. Evidence is also provided that, on average, out-of-frame dystrophin exons have significantly stronger CVs and more favorable DeltaDG degrees 37 values; this feature has never been reported and might reflect evolutionary-driven minimization of out-of-frame exon misplicing. Dystrophin splicing mutations have been reported to determine either Duchenne or Becker Muscular Dystrophy, but no comprehensive genotypic/phenotypic correlation has ever been investigated. We have analyzed splicing affecting single base-pair substitutions in the dystrophin gene with respect to their effect on splicing parameters; functional and clinical consequences are also reported. We have found 5'-splice-site mutation occurrence to be statistically related to mutability quotients and propose the use of DeltaDG degrees 37 values as a more effective tool than CV alone to describe donor site mutation consequences. Our analysis also indicates a nearly 100% correlation between clinical phenotype and the reading-frame rule determined at the RNA level. We consider that elucidation of the relative importance of splicing determinants might help to clarify the molecular mechanisms that direct correct splicing in complex genes and might be useful in the validation of predictive models.

Dystrophin↗

PubMed1999

Steroid 21-hydroxylase deficiency: mutational spectrum in Denmark, three novel mutations, and in vitro expression analysis.

We have investigated 68 unrelated 21-hydroxylase deficient Danish patients, representing 136 alleles, and determined the mutational spectrum of the CYP21 gene. The most frequent mutations detected were deletion of CYP21 and the splice mutation in intron 2 (I2-splice). Segregation analysis showed evidence of a de novo mutation in each of two patients. Three novel mutations were detected: G64E in exon 1, Q262X in exon 7, and A362V in exon 8. G64E and A362V were introduced in the CYP21 cDNA by in vitro site-directed mutagenesis, and the two corresponding proteins were transiently expressed in COS-7 cells. The activity of 21-hydroxylase was determined using the two hormone substrates 17-hydroxyprogesterone and progesterone. The analysis showed no enzyme activity for any of the substrates, a result that correlates well with the severity of the patients' disease.

Adrenal Hyperplasia, Congenital↗

PubMed1999

Functional changes in troponin T by a splice donor site mutation that causes hypertrophic cardiomyopathy.

A splice donor site mutation in intron 15 of the cardiac troponin T (TnT) gene has been shown to cause familial hypertrophic cardiomyopathy (HCM). In this study, two truncated human cardiac TnTs expected to be produced by this mutation were expressed in Escherichia coli and partially (50-55%) exchanged into rabbit permeabilized cardiac muscle fibers. The fibers into which a short truncated TnT, which lacked the COOH-terminal 21 amino acids because of the replacement of 28 amino acids with 7 novel residues, had been exchanged generated a Ca(2+)-activated maximum force that was slightly, but statistically significantly, lower than that generated by fibers into which wild-type TnT had been exchanged when troponin I (TnI) was phosphorylated by cAMP-dependent protein kinase. A long truncated TnT simply lacking the COOH-terminal 14 amino acids had no significant effect on the maximum force-generating capability in the fibers with either phosphorylated or dephosphorylated TnI. Both these two truncated TnTs conferred a lower cooperativity and a higher Ca(2+) sensitivity on the Ca(2+)-activated force generation than did wild-type TnT, independent of the phosphorylation of TnI by cAMP-dependent protein kinase. The results demonstrate that the splice donor site mutation in the cardiac TnT gene impairs the regulatory function of the TnT molecule, leading to an increase in the Ca(2+) sensitivity, and a decrease in the cooperativity, of cardiac muscle contraction, which might be involved in the pathogenesis of HCM.

Animals↗

PubMed1995

Cardiac myosin binding protein-C gene splice acceptor site mutation is associated with familial hypertrophic cardiomyopathy.

Familial hypertrophic cardiomyopathy (FHC) is an autosomal dominant disease characterized by a ventricular hypertrophy predominantly affecting the interventricular septum and associated with a large extent of myocardial and myofibrillar disarray. It is the most common cause of sudden death in the young. In the four disease loci found, three genes have been identified which code for beta-myosin heavy chain, cardiac troponin T and alpha-tropomyosin. Recently the human cardiac myosin binding protein-C (MyBP-C) gene was mapped to chromosome 11p11.2 (ref. 8), making this gene a good candidate for the fourth locus, CMH4 (ref. 5). Indeed, MyBP-C is a substantial component of the myofibrils that interacts with several proteins of the thick filament of the sarcomere. In two unrelated French families linked to CMH4, we found a mutation in a splice acceptor site of the MyBP-C gene, which causes the skipping of the associated exon and could produce truncated cardiac MyBP-Cs. Mutations in the cardiac MyBP-C gene likely cause chromosome 11-linked hypertrophic cardiomyopathy, further supporting the hypothesis that hypertrophic cardiomyopathy results from mutations in genes encoding contractile proteins.

Amino Acid Sequence↗