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At least 289 records · Page 16Linked to original sources
PubMed2003

Analysis of the consequences of premature termination codons within factor VIII coding sequences.

Inhibitor antibody formation is a complication of factor VIII (FVIII) replacement therapy due to a failure to synthesize sufficient FVIII protein to induce immune tolerance. The incidence of nonsense mutations in inhibitor patients is high, however, this association is variable according to the position of the mutation. We have studied the effect of nonsense mutations on accumulation of FVIII mRNA, protein translation and secretion. Appropriately processed mRNA was detected in cells transfected with wild-type R1966X and R2116X expression constructs and no evidence of nonsense-mediated decay was observed. All constructs directed the translation of detectable intracellular FVIII antigen, however, secreted FVIII was detected only in conditioned media of cells transfected with wild-type cDNA. We have also analyzed ectopic FVIII mRNA transcripts in the lymphocytes of six hemophilia A patients with nonsense mutations (Q139X, R583X, R1941X, R1966X and two unrelated patients with R2116X). FVIII mRNA was detectable in every case. In R1941X and R1966X only normally spliced transcripts were present. In Q139X, R583X and R2116X aberrantly spliced transcripts were observed with two distinct patterns in two individuals with the R2116X mutation. No correlation between mutation, transcript pattern and incidence of inhibitor development was apparent.

Alternative Splicing↗
PubMed1999

Expression of a new isoform of the tumor susceptibility TSG101 protein lacking a leucine zipper domain in Burkitt lymphoma cell lines.

The tumor susceptibility gene, TSG101, has been identified as a candidate tumor suppressor gene. We have examined the expression of TSG101 in Burkitt lymphoma cell lines. Several aberrant messages were detected in all cell lines. Aberrant splice donor sites are located within exon 1 at positions 132, 154, 172 and 284. Splice acceptors are located at positions 847 and 1054 within exon 5. The aberrant messages are coexpressed with a normal message and could be the result of additional splicing reactions of the mature message that behaves as an intermediate. The normal message codes for 46 kDa protein (TSG101A). One aberrant message joins in frame nucleotides 283-1055 and codes for a protein isoform of 17 kDa (TSG101B), as demonstrated by in vitro translation assays. The TSG101B isoform lacks the leucine zipper near the C-terminus, a transcriptional repressor domain, and retains most of the N-terminal region which has homology to E2 ubiquitin regulatory enzymes and the CROC-1 transcriptional regulator. The TSG101B isoform was detected in sixteen out of twenty-two (72%) BL cell lines, but not in normal lymphoid populations. The presence of two TSG101 isoforms with different dimerization potential opens up a new level of regulation of the TSG101 proteins possibly affecting cell cycle regulation.

Amino Acid Sequence↗
PubMed1998

A novel splice site mutation of the EXT2 gene in a Finnish hereditary multiple exostoses family. Mutations in brief no. 197. Online.

Hereditary multiple exostoses is a dominantly inherited disease characterized by multiple benign osteochondromas. The affected individuals have an increased risk of developing sarcoma. A large Finnish family with hereditary multiple exostosis was analyzed to find the disease-causing mutation. Blood samples were obtained from 35 family members, including 21 affected and 14 unaffected individuals. Using 2-point linkage analysis the EXT phenotype was shown to be linked to the recently cloned EXT2 gene on chromosome 11p11. The coding region of the gene was sequenced and a previously unreported splice site mutation found. This G to T transversion within a 5-prime splice donor site following exon 6 was shown to cause aberrant splicing of RNA. The described change is considered to be a novel disease-causing mutation in the EXT2 gene.

Alternative Splicing↗
PubMed1995

A splicing mutation, a nonsense mutation (Y167X) and two missense mutations (I159T and A209V) in Spanish patients with ornithine transcarbamylase deficiency.

Four novel mutations are identified in the ornithine transcarbamylase (OTC) gene, in four patients with OTC deficiency (an X-linked disorder). The mutations represent three different categories: missense (Ile159Thr and Ala209Val), nonsense (Tyr167Stop), and causing inefficient splicing (G-->A in the first intronic base) with associated aberrant splicing. They are located in exons 5, and 6, and in intron 3. Two of the mutations arose de novo in the patients, and only one mutation occurs at a CpG site. The nonsense and the splicing mutation cause, respectively, lethal early onset and non-lethal, delayed early onset clinical presentations in males. Our results confirm for Spain the high genotypic heterogeneity of OTC deficiency.

Base Sequence↗
PubMed2000

Hereditary spastic paraplegia caused by mutations in the SPG4 gene.

Autosomal dominant hereditary spastic paraplegia (AD-HSP) is a genetically heterogeneous neurodegenerative disorder characterised by progressive spasticity of the lower limbs. The SPG4 locus at 2p21-p22 accounts for 40-50% of all AD-HSP families. The SPG4 gene was recently identified. It is ubiquitously expressed in adult and foetal tissues and encodes spastin, an ATPase of the AAA family. We have now identified four novel SPG4 mutations in German AD-HSP families, including one large family for which anticipation had been proposed. Mutations include one frame-shift and one missense mutation, both affecting the Walker motif B. Two further mutations affect two donor splice sites in introns 12 and 16, respectively. RT-PCR analysis of both donor splice site mutations revealed exon skipping and reduced stability of aberrantly spliced SPG4 mRNA. All mutations are predicted to cause loss of functional protein. In conclusion, we confirm in German families that SPG4 mutations cause AD-HSP. Our data suggest that SPG4 mutations exert their dominant effect not by gain of function but by haploinsufficiency. If a threshold level of spastin were critical for axonal preservation, such threshold dosage effects might explain the variable expressivity and incomplete penetrance of SPG4-linked AD-HSP.

Adenosine Triphosphatases↗
PubMed2000

Report of five novel and one recurrent COL2A1 mutations with analysis of genotype-phenotype correlation in patients with a lethal type II collagen disorder.

Achondrogenesis II-hypochondrogenesis and severe spondyloepiphyseal dysplasia congenita (SEDC) are lethal forms of dwarfism caused by dominant mutations in the type II collagen gene (COL2A1). To identify the underlying defect in seven cases with this group of conditions, we used the combined strategy of cartilage protein analysis and COL2A1 mutation analysis. Overmodified type II collagen and the presence of type I collagen was found in the cartilage matrix of all seven cases. Five patients were heterozygous for a nucleotide change that predicted a glycine substitution in the triple helical domain (G313S, G517V, G571A, G910C, G943S). In all five cases, analysis of cartilage type II collagen suggested incorporation of the abnormal alpha1(II) chain in the extracellular collagen trimers. The G943S mutation has been reported previously in another unrelated patient with a strikingly similar phenotype, illustrating the possible specific effect of the mutation. The radiographically less severely affected patient was heterozygous for a 4 bp deletion in the splice donor site of intron 35, likely to result in aberrant splicing. One case was shown to be heterozygous for a single nucleotide change predicted to result in a T1191N substitution in the carboxy-propeptide of the proalpha1(II) collagen chain. Study of the clinical, radiographic, and morphological features of the seven cases supports evidence for a phenotypic continuum between achondrogenesis II-hypochondrogenesis and lethal SEDC and suggests a relationship between the amount of type I collagen in the cartilage and the severity of the phenotype.

Achondroplasia↗
PubMed2006

Cryptic splice site usage in exon 7 of the human fibrinogen Bbeta-chain gene is regulated by a naturally silent SF2/ASF binding site within this exon.

In this work we report the identification of a strong SF2/ASF binding site within exon 7 of the human fibrinogen Bbeta-chain gene (FGB). Its disruption in the wild-type context has no effect on exon recognition. However, when the mutation IVS7 + 1G>T--initially described in a patient suffering from congenital afibrinogenemia--is present, this SF2/ASF binding site is critical for cryptic 5'ss (splice site) definition. These findings, besides confirming and extending previous results regarding the effect of SF2/ASF on cryptic splice site activation, identify for the first time an enhancer sequence in the FGB gene specific for cryptic splice site usage. Taken together, they suggest the existence of a splicing-regulatory network that is normally silent in the FGB natural splicing environment but which can nonetheless influence splicing decisions when local contexts allow. On a more general note, our conclusions have implications for the evolution of alternative splicing processes and for the development of methods to control aberrant splicing in the context of disease-causing mutations.

Base Sequence↗
PubMed2000

Jagged1 (JAG1) mutation detection in an Australian Alagille syndrome population.

Alagille syndrome (AGS) is an autosomal dominant disorder characterized by abnormal development of the liver, heart, skeleton, eye, and face. Mutations in the Jagged1 gene (JAG1) have been found to result in the AGS phenotype and both protein truncating mutations and missense mutations have been identified. Using single stranded conformational polymorphism analysis we have screened 22 AGS affected individuals from 19 families for mutations within Jagged1. Twelve distinct Jagged1 mutations were identified in 15 (68.2%) of the 22 AGS cases, seven of which are novel. The mutations include three small deletions (25%), two small insertions (16.6%), three missense mutations (25%), two nonsense mutations (16.6%), and two splice-site mutations (16.6%). These mutations are spread across the entire coding sequence of the gene and most are localized to highly conserved motifs of the protein predicted to be important for Jagged1 function. One-half of the mutations found in this study are located between exons 9 and 12, a region constituting only 12% of the coding sequence. A splice-donor site mutation in intron 11 was shown to cause aberrant splicing of Jagged1 mRNA, consequently terminating translation prematurely in exon 12. The results of this study are consistent with the proposal that either haploinsufficiency for wild type Jagged1 and/or dominant negative effects produced by mutated Jagged1 are responsible for the AGS phenotype.

Alagille Syndrome↗
PubMed2004

Adult-onset ataxia telangiectasia due to ATM 5762ins137 mutation homozygosity.

Ataxia telangiectasia (A-T) is an autosomal recessive neurodegenerative disorder that arises because of mutations in the ATM gene. The 5762ins137 A-->G point mutation activates a cryptic splice donor site resulting in a 137 bp intronic insert being aberrantly spliced into the ATM transcript. However, normal ATM transcript also is produced from this affected allele, albeit at significantly reduced levels. An exceptionally mild A-T phenotype occurs as a result of homozygosity for the 5762ins137 mutation because of relative preservation of ATM protein expression/kinase activity.

Adult↗
PubMed2006

DHPLC-based mutation analysis of ENG and ALK-1 genes in HHT Italian population.

Hereditary haemorrhagic telangiectasia (HHT or Rendu-Osler-Weber syndrome) is an autosomal dominant disorder characterized by localized angiodysplasia due to mutations in endoglin, ALK-1 gene, and a still unidentified locus. The lack of highly recurrent mutations, locus heterogeneity, and the presence of mutations in almost all coding exons of the two genes makes the screening for mutations time-consuming and costly. In the present study, we developed a DHPLC-based protocol for mutation detection in ALK1 and ENG genes through retrospective analysis of known sequence variants, 20 causative mutations and 11 polymorphisms, and a prospective analysis on 47 probands with unknown mutation. Overall DHPLC analysis identified the causative mutation in 61 out 66 DNA samples (92.4%). We found 31 different mutations in the ALK1 gene, of which 15 are novel, and 20, of which 12 are novel, in the ENG gene, thus providing for the first time the mutational spectrum in a cohort of Italian HHT patients. In addition, we characterized the splicing pattern of ALK1 gene in lymphoblastoid cells, both in normal controls and in two individuals carrying a mutation in the non-invariant -3 position of the acceptor splice site upstream exon 6 (c.626-3C>G). Functional essay demonstrated the existence, also in normal individuals, of a small proportion of ALK1 alternative splicing, due to exon 5 skipping, and the presence of further aberrant splicing isoforms in the individuals carrying the c.626-3C>G mutation.

Activin Receptors, Type II↗
PubMed1991

Muscle-specific accumulation of Drosophila myosin heavy chains: a splicing mutation in an alternative exon results in an isoform substitution.

We show that the molecular lesions in two homozygousviable mutants of the Drosophila muscle myosin heavy chain gene affect an alternative exon (exon 9a) which encodes a portion of the myosin head that is highly conserved among both cytoplasmic and muscle myosins of all organisms. In situ hybridization and Northern blotting analysis in wild-type organisms indicates that exon 9a is used in indirect flight muscles whereas both exons 9a and 9b are utilized in jump muscles. Alternative exons 9b and 9c are used in other larval and adult muscles. One of the mutations in exon 9a is a nonsense allele that greatly reduces myosin RNA stability. It prevents thick filament accumulation in indirect flight muscles and severely reduces the number of thick filaments in a subset of cells of the jump muscles. The second mutation affects the 5' splice site of exon 9a. This results in production of an aberrantly spliced transcript in indirect flight muscles, which prevents thick filament accumulation. Jump muscles of this mutant substitute exon 9b for exon 9a and consequently have normal levels of thick filaments in this muscle type. This isoform substitution does not obviously affect the ultrastructure or function of the jump muscle. Analysis of this mutant illustrates that indirect flight muscles and jump muscles utilize different mechanisms for alternative RNA splicing.

Amino Acid Sequence↗
PubMed1998

Nine novel germline mutations of STK11 in ten families with Peutz-Jeghers syndrome.

Peutz-Jeghers Syndrome (PJS) is an autosomal dominant hereditary disease characterized by hamartomatous polyposis involving the entire bowel. Recently STK11, a gene bearing a mutation responsible for PJS, was isolated. We investigated the entire coding region of STK11 in 15 unrelated PJS families by the PCR-SSCP (polymerase chain reaction-single strand conformation polymorphism) method and PCR-direct sequence analysis, and found nine different, novel mutations among ten of those families. One nonsense mutation and five different frameshift mutations (two families carried the same mutation), all of which would cause truncation of the gene product, were found in seven families; mutations found in five families were clustered within exon 6. Among these five mutations, three occurred at the mononucleotide-repeat region (CCCCCC) of codons 279-281, suggesting that this region is likely to be a mutational hotspot of this gene. One of the remaining three families carried a 3-bp in-frame deletion that would eliminate an asparagine residue within a kinase domain of the product; the other two carried intronic mutations at or adjacent to the consensus dinucleotide sequences of splice-acceptor or -donor sites, which were likely to lead to aberrant splicing.

AMP-Activated Protein Kinase Kinases↗
PubMed2003

Low frequency of FAS mutations in Reed-Sternberg cells of Hodgkin's lymphoma.

Reed-Sternberg (RS) cells, the neoplastic elements of Hodgkin's lymphoma (HL), usually lack B-cell receptor expression. Normal germinal center B cells, with lack of or low-affinity B-cell receptor expression, are eliminated via FAS-induced apoptosis. RS cells express FAS, but are rescued from apoptosis by a transforming event. It is known that HL-derived cell lines are resistant to FAS-mediated apoptosis. To investigate potential causes for this resistance, FAS mutations and c-FLIP expression were studied in four HL-derived cell lines and 20 cases of HL. L1236 was found to have a splice donor site mutation in intron 7 that resulted in an aberrantly spliced FAS transcript. Screening of microdissected RS cells revealed loss of heterozygosity for a known exon 7 polymorphism in two of six informative cases indicating loss of one FAS allele. In one of the two cases with loss of heterozygosity a hemizygous mutation was detected in exon 9. c-FLIP expression was observed in all HL cell lines and in RS cells of all HL cases. Our data show that FAS mutations are rare and suggest that overexpression of c-FLIP, which was present in all cases, is involved in the resistance to FAS-mediated apoptosis.

Apoptosis↗
PubMed2003

Loss of ligand-binding specificity of fibroblast growth factor receptor 2 by RNA splicing in human chondrosarcoma cells.

We have previously reported the alternatively spliced transcripts of fibroblast growth factor receptor (FGFR) 3 derived by aberrant splicing in human cancers. Here, we describe a novel splice variant of FGFR2 (FGFR2DeltaIII) arising from skipping exons 7-10, resulting in the deletion of Ig-like-III domain in human chondrosarcoma cell. Sf9 cells expressing FGFR2DeltaIII were able to bind FGF1, FGF2, and FGF7, leading to loss of ligand-binding specificity. Together with our previous findings, the present studies suggest that mRNA splicing plays an important role in the regulation of FGFRs' function.

Blotting, Western↗
PubMed2003

Oligosymptomatic cornea verticillata in a heterozygote for Fabry disease: a novel mutation in the alpha-galactosidase gene.

BACKGROUND: Fabry disease is an X-linked genetic disorder involving sphingolipid catabolism, which is caused by lysosomal alpha-galactosidase A deficiency. Ophthalmological findings such as corneal and lens opacities and conjunctival and retinal vessel abnormalities can be the only and/or the first recognizable symptoms, especially in heterozygous females. METHODS: We report on a 34-year-old German woman with cornea verticillata. The alpha-galactosidase A activity was determined in leukocytes using a fluorescence substrate, and the sequence analysis of the alpha galactosidase A gene was performed with genomic DNA. RESULTS: The alpha-galactosidase A activity in leukocytes was significantly low (0.24 nmol/min/mg protein; normal range, 0.4-1.2), which is compatible with a heterozygote for Fabry disease. The following sequence analysis revealed a heterozygous transition in position IVS5 + 2 T > C. Transition of thymine (T) to cytosine (C) affects the donor splice motive of exon 5 and most probably leads to an aberrant splicing procedure of the alpha-galactosidase A gene. CONCLUSION: Our case emphasizes the importance of ophthalmological findings in Fabry disease. The subsequent biochemical and molecular analysis provides a secure diagnosis of female carriers of Fabry disease.

Adult↗
PubMed1995

Deletion of the 3' splice site of the leader-variable region intron of immunoglobulin heavy chain genes induces a direct splicing of leader to constant region, resulting in the production of truncated mu-chains.

An Abelson virus-transformed immature B cell line, AT8-1-12-5-2, produced truncated mu-chains. Sequencing analysis of the VHDJH complex on the expressed H-chain allele revealed the deletion of 75 nucleotides that involved leader-variable region intron and the 5' end of the variable region, which resulted in the loss of the 3' splice site of leader-variable region intron. Sequence studies of a leader- and CH1-containing cDNA clone showed that leader region was directly spliced to the CH1 exon, resulting in the production of the truncated mu-chains without variable portion. Our results demonstrated for the first time that the only loss of the 3' splice site of leader-variable region intron could induce an aberrant splicing between leader and constant region.

Abelson murine leukemia virus↗
PubMed1999

Variable levels of normal RNA in different fetal organs carrying a cystic fibrosis transmembrane conductance regulator splicing mutation.

Disease severity varies among cystic fibrosis (CF) patients carrying the same cystic fibrosis transmembrane conductance regulator (CFTR) genotype and among organs of the same individual. It has been shown that the class V splicing mutation 3849 + 10 kb C--> T produces both normal and aberrantly spliced CFTR transcripts. We analyzed the levels of normal CFTR messenger RNA (mRNA) in different organs of an aborted fetus carrying the 3849 + 10 kb C--> T mutation, and found that they correlated with the histopathologic changes observed in these organs. We performed semiquantitative nondifferential reverse transcription-polymerase chain reaction on several organs from a 22-wk aborted CF fetus carrying the 3849 + 10 kb C--> T mutation. A very low level (1%) of normal CFTR mRNA was detected in the severely affected ileum of this fetus. Higher levels were found in the histopathologically unaffected trachea (17%), colon (19%), and lung (26%). Thus, as early as in utero, the regulation of alternative splice-site selection is an important mechanism underlying variable CF severity. Understanding of the mechanisms regulating alternative splicing in different tissues will contribute to potential therapy for patients carrying splicing mutations in CF and other human disease genes.

Alternative Splicing↗
PubMed1998

Splice donor site mutations in the 3-hydroxy-3-methylglutaryl coenzyme A reductase gene cause a deficiency of the endoplasmic reticulum 3-hydroxy-3-methylglutaryl coenzyme A reductase protein in UT2 cells.

UT2 cells are a mutant clone of Chinese hamster ovary (CHO) cells that are deficient in the 97 kDa endoplasmic reticulum (ER) 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase protein. The analysis of UT2 cell cDNA and genomic DNA has led to the identification of two novel point mutations in intronic sequences of the ER HMG-CoA reductase gene. One mutation identified at the +1 position (G --> A) of the 5' splice site of exon 11-12 junction was shown to cause exon 11 skipping which resulted in the insertion of premature stop codons. We also identified a second mutation at the +5 position (G --> A) of the 5' splice site in the intron spanning exons 13 and 14. Furthermore, the data indicate that the two mutations in the reductase gene are present on the same allele. As demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) of UT2 cell mRNA, the mutations produce aberrant spliced messages. If the aberrant messages were translated, truncated proteins of 44 kDa or 66 kDa would be predicted. More importantly, these truncated proteins would be expected not to have catalytic activity. In addition, we have also recently demonstrated that the UT2 cells express a 90 kDa HMG-CoA reductase protein that is localized exclusively in peroxisomes, and is up-regulated when the cells are grown in the absence of added mevalonate. Thus, the mutations identified in the ER reductase gene in UT2 cells indicate that neither a 97 kDa nor a 90 kDa reductase protein can be produced from this gene.

Alleles↗