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Results for “Splicing mutations”

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PubMed1998

New mutations in the KVLQT1 potassium channel that cause long-QT syndrome.

BACKGROUND: Long-QT syndrome (LQTS) is an inherited cardiac arrhythmia that causes sudden death in young, otherwise healthy people. Four genes for LQTS have been mapped to chromosome 11p15.5 (LQT1), 7q35-36 (LQT2), 3p21-24 (LQT3), and 4q25-27 (LQT4). Genes responsible for LQT1, LQT2, and LQT3 have been identified as cardiac potassium channel genes (KVLQT1, HERG) and the cardiac sodium channel gene (SCN5A). METHODS AND RESULTS: After studying 115 families with LQTS, we used single-strand conformation polymorphism (SSCP) and DNA sequence analysis to identify mutations in the cardiac potassium channel gene, KVLQT1. Affected members of seven LQTS families were found to have new, previously unidentified mutations, including two identical missense mutations, four identical splicing mutations, and one 3-bp deletion. An identical splicing mutation was identified in affected members of four unrelated families (one Italian, one Irish, and two American), leading to an alternatively spliced form of KVLQT1. The 3-bp deletion arose de novo and occurs at an exon-intron boundary. This results in a single base deletion in the KVLQT1 cDNA sequence and alters splicing, leading to the truncation of KVLQT1 protein. CONCLUSIONS: We have identified LQTS-causing mutations of KVLQT1 in seven families. Five KVLQT1 mutations cause the truncation of KVLQT1 protein. These data further confirm that KVLQT1 mutations cause LQTS. The location and character of these mutations expand the types of mutation, confirm a mutational hot spot, and suggest that they act through a loss-of-function mechanism or a dominant-negative mechanism.

Case-Control Studies↗

PubMed1998

Prenatal diagnosis of steroid 21-hydroxylase deficiency by the modified polymerase chain reaction to detect splice site mutation in the CYP21 gene.

A splicing junction mutation at nucleotide 656 (A-> G substitution, I2G) in the steroid 21-hydroxylase gene (CYP21) is the most frequently detected mutation in patients with the salt-wasting and simple-virilizing forms of steroid 21-hydroxylase deficiency (approximately 60%). In this disease, prenatal diagnosis and treatment to minimize the effects of excess androgen in affected females has been advocated. Therefore, to detect the I2G mutation rapidly, accurately, and without the use of radioisotope, we developed a modified polymerase chain reaction (PCR) with a mismatched 3' nucleotide primer to introduce a new restriction site upon PCR amplification of the mutant allele. This allowed the mutant allele to be identified readily by restriction enzyme digestion of the PCR product, and subsequently this PCR product was subjected to restriction enzyme digestion for diagnosis. Chorionic villus biopsy samples (CVS) were obtained at 10 to 11 weeks gestation from two females carrying fetuses at risk for steroid 21-hydroxylase deficiency. Prenatal diagnosis was successful in both cases. One affected female was treated with dexamethasone to term. In the other case, treatment was withdrawn at an early stage when testing revealed a normal fetus. The results demonstrate the rapid and accurate detection of the I2G mutation by this method, thereby indicating the feasibility of for prenatal diagnosis of the I2G mutation.

Adrenal Hyperplasia, Congenital↗

PubMed2000

Illegitimate splicing of the NF1 gene in healthy individuals mimics mutation-induced splicing alterations in NF1 patients.

Neurofibromatosis type 1 (NF1) is a common inherited disease affecting one in 3,500 individuals. The mutation rate in the NF1 gene is one of the highest known for human genes. Compared to other methods, the protein truncation test (PTT) and subsequent sequence analysis of cloned cDNA provides improved efficiency in detecting NF1 mutations that are dispersed throughout the gene spanning 350 kb of genomic DNA. Sequencing of cDNA of patients affected with NF1 mutations revealed multiple splicing errors. Since similar missplicings were also found in "aged" blood of healthy individuals, they are most likely attributable to a general decrease in splice site selection in aged blood. We show that restoring viability of lymphocytes before RNA extraction by cultivation and PHA stimulation diminishes aberrant splicing in aged blood and is thus useful to circumvent splicing alterations which are frequently compromising mutation detection in patient samples and mimic mutation-induced alterations of mRNA.

Cellular Senescence↗

PubMed1999

Group II intron splicing in Escherichia coli: phenotypes of cis-acting mutations resemble splicing defects observed in organelle RNA processing.

The mitochondrial group IIB intron rI1, from the green algae Scenedesmus obliquus ' LSUrRNA gene, has been introduced into the lacZ gene encoding beta-galacto-sidase. After DNA-mediated transformation of the recombinant lacZ gene into Escherichia coli, we observed correct splicing of the chimeric precursor RNA in vivo. In contrast to autocatalytic in vitro self-splicing, intron processing in vivo is independent of the growth temperature, suggesting that in E.coli, trans -acting factors are involved in group II intron splicing. Such a system would seem suitable as a model for analyzing intron processing in a prokaryotic host. In order to study further the effect of cis -mutations on intron splicing, different rI1 mutants were analyzed (with respect to their splicing activity) in E.coli. Although the phenotypes of these E. coli intron splicing mutants were identical to those which can be observed during organellar splicing of rI1, they are different to those observed in in vitro self-splicing experiments. Therefore, in both organelles and prokaryotes, it is likely that either similar splicing factors or trans -acting factors exhibiting similar functions are involved in splicing. We speculate that ubiquitous trans -acting factors, via recent horizontal transfer, have contributed to the spread of group II introns.

Alternative Splicing↗

PubMed1996

Hereditary tyrosinemia type 1: novel missense, nonsense and splice consensus mutations in the human fumarylacetoacetate hydrolase gene; variability of the genotype-phenotype relationship.

The complete fumarylacetoacetate hydrolase (FAH) genotype of probands of thirteen unrelated families with hereditary tyrosinemia type 1 (HT 1) was established. The screening was performed by analysis of exons 2-14 of the FAH gene by using the polymerase chain reaction (PCR) and of the mRNA by reverse transcription/PCR. Nine different mutations were identified, of which six are novel. Three mutations involve consensus sequences for correct splicing, viz. IVS 6-1 (g-t), IVS 7-1 (g-a) and IVS 12 + 5 (g-a). Two missense mutations (C193R and G369V) and three nonsense mutations (R237X, E357X and E364X) were found. One silent mutation N232N was associated with the skipping of exon 8 from the FAH mRNA. Analysis of the effect of the respective mutations on the FAH mRNA showed a strong reduction of FAH mRNA levels in association with the nonsense mutations, and normal levels with the missense mutations. The splice consensus mutations give deletions of complete or small parts of exon sequences from the FAH mRNA. Data suggest a founder effect for several of the mutations, with a frequency for both the IVS 6-1 (g-t) and IVS 12 + 5 (g-a) mutations of approximately 30% in the HT 1 probands. No strict correlation between genotype and phenotype, i.e. the acute, subacute or chronic form of HT 1, was evident.

Alleles↗

PubMed2002

Human papillomavirus type 31 replication modes during the early phases of the viral life cycle depend on transcriptional and posttranscriptional regulation of E1 and E2 expression.

The E1 and E2 proteins are both required for papillomavirus DNA replication, and replication efficiency is controlled by the abundance of these factors. In human papillomaviruses (HPVs), the regulation of E1 and E2 expression and its effect on viral replication are not well understood. In particular, it is not known if E1 and E2 modulate their own expression and how posttranscriptional mechanisms may affect the levels of the replication proteins. Previous studies have implicated splicing within the E6 open reading frame (ORF) as being important for modulating replication of HPV type 31 (HPV31) through altered expression of E1 and E2. To analyze the function of the E6 intron in viral replication more specifically, we examined the effects of E6 splicing mutations in the context of entire viral genomes in transient assays. HPV31 genomes which had mutations in the splice donor site (E6SD) or the splice acceptor site (E6SA), a deletion of the intron (E6ID), or substituted heterologous intron sequences (E6IS) were constructed. Compared to wild-type (wt) HPV31, pHPV31-E6SD, -E6SA, and -E6IS replicated inefficiently while pHPV31-E6ID replicated at an intermediate level. Cotransfection of the E6 mutant genomes with an E1 expression vector strongly activated their replication levels, indicating that efficient expression of E1 requires E6 internal splicing. In contrast, replication was activated only moderately with an E2 expression vector. Replacing the wt E6 intron in HPV31 with a heterologous intron from simian virus 40 (E6SR2) resulted in replication levels similar to that of the wt in the absence of expression vectors, suggesting that mRNA splicing upstream of the E1 ORF is important for high-level replication. To examine the effects of E6 intron splicing on E1 and E2 expression directly, we constructed reporter DNAs in which the luciferase coding sequences were fused in frame to the E1 (E1Luc) or E2 (E2Luc) gene. Reporter activities were then analyzed in transient assays with cotransfected E1 or E2 expression vectors. Both reporters were moderately activated by E1 in a dose-dependent manner. In addition, E1Luc was activated by low doses of E2 but was repressed at high doses. In contrast, E2 had little effect on E2Luc activity. These data indicate that E1 expression and that of E2 are interdependent and regulated differentially. When the E6 splicing mutations were analyzed in both reporter backgrounds, only E1Luc activities correlated with splicing competence in the E6 ORF. These findings support the hypothesis that the E6 intron primarily regulates expression of E1. Finally, in long-term replication assays, none of the E6 mutant genomes could be stably maintained. However, cotransfection of the E6 splicing mutant genomes with pHPV31-E7NS, which contains a nonsense mutation in the E7 coding sequence, restored stable replication of some mutants. Our observations indicate that E1 expression and that of E2 are differentially regulated at multiple levels and that efficient expression of E1 is required for transient and stable viral replication. These regulatory mechanisms likely act to control HPV copy number during the various phases of the viral life cycle.

Carcinoma, Squamous Cell↗

PubMed1999

A Japanese case with Frasier syndrome caused by the splice junction mutation of WT1 gene.

The Wilms' tumor suppressor gene, WT1, plays an important role in the development of the urogenital system and also subsequent normal function of this system. Recently, the splice mutations in intron 9 of WT1 gene have been detected in Frasier syndrome, which is characterized by streak gonads, pseudohermaphroditism, slowly progressive nephropathy and frequent development of gonadoblastoma. Here to elucidate the molecular basis in a Japanese patient of Frasier syndrome, WT1 gene was analyzed by polymerase-chain-reaction (PCR) and direct sequencing. We identified the splice junction mutation in intron 9 of WT1, which is recognized as a mutation hot-spot in intron 9. This finding concludes that 1) the mutation in intron 9 might be the cause of Frasier syndrome, and 2) the mutation hot-spot in Japanese and Caucasian patients is similar.

Adolescent↗

PubMed1999

5' splice site mutations in tau associated with the inherited dementia FTDP-17 affect a stem-loop structure that regulates alternative splicing of exon 10.

Missense and splice site mutations in the microtubule-associated protein tau gene were recently found associated with fronto-temporal dementia and parkinsonism linked to chromosome 17 (Poorkaj et al. (1998) Ann. Neurol. 43, 815-825; Hutton et al. (1998) Nature 393, 702-705; Spillantini et al. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 7737-7741). The mutations in the 5' splice site of exon 10 were shown to increase the ratio of tau mRNAs containing exon 10 and thus the proportion of Tau protein isoforms with 4 microtubule binding repeat domains, although how this increase leads to neurodegeneration is presently unclear. The mechanism by which these mutations increase tau exon 10 splicing was not determined, although the mutations were predicted to disrupt a potential stem-loop structure that was likely involved in the regulation of exon 10 alternative splicing. Here we describe in vitro splicing assays and RNA structural analysis that demonstrate that the mutations do indeed act through disruption of the stem-loop structure and that the stability of this secondary structure feature at least partially determines the ratio of tau exon 10+/- transcripts. In addition, we provide evidence that the stability of the stem-loop structure underlies the alternative splicing of this exon in other species.

Alternative Splicing↗

PubMed1998

Suppression of the 5' splice site mutation in the Nagase analbuminemic rat with mutated U1snRNA.

Nagase analbuminemic rats (NAR) are deficient in serum albumin due to skipping of the albumin exon H caused by a mutation in the intron HI. This mutation deletes nucleotides from +5 to +11 in the 5' splice site region, where it interacts with U1snRNA. To determine whether the mutation can be suppressed by the compensatory base substitution in U1snRNA, we constructed mutated U1snRNA genes with various degrees of complementarity to the mutated 5' splice site. Several mutated U1snRNA genes activated the mutated 5' splice site of the intron HI, when cotransfected with the albumin minigene derived from NAR. In vivo activity of these mutated U1snRNAs correlated well with the predicted thermodynamic stability. Since mutation in the 5' splice site is one of common causes of genetic defects in human (5), these data indicate that NAR is a good model system to examine the possibility of gene therapy using a mutated U1snRNA.

Animals↗

PubMed2005

High frequency of the 425A-->G splice-site mutation and novel mutations of the COL7A1 gene in central Europe: significance for future mutation detection strategies in dystrophic epidermolysis bullosa.

BACKGROUND: Mutations in the type VII collagen gene (COL7A1) are responsible for dominant and recessive forms of dystrophic epidermolysis bullosa (DEB). These mutations are usually specific for individual families; only a few cases of recurring mutations have been identified. OBJECTIVES: Forty-three unrelated Hungarian and German patients with different DEB phenotypes were screened for novel and recurrent COL7A1 mutations. METHODS: All patients were classified based on clinical and genetic findings, skin immunofluorescent antigen mapping, and electron microscopic studies. Mutation analysis was performed by amplification of genomic DNA with polymerase chain reaction using COL7A1-specific primers, heteroduplex analysis, and direct nucleotide sequencing. Restriction endonuclease digestion was used for family screening and mutation verification. Results In this group of patients, the splice-site mutation 425A-->G was observed frequently, in 11 of 86 alleles (12.8%), once in homozygous form and in nine cases in heterozygous form. One of 100 control alleles from clinically unaffected individuals also carried the mutation. We also identified three novel mutations: the 976-3C-->A splice-site mutation, and the 4929delT and 8441-15del20 deletions. CONCLUSIONS: High recurrence of the splice-site mutation 425A-->G in central European patients with DEB should be taken into account when designing COL7A1 mutation detection strategies. Reporting of three novel COL7A1 mutations in this study further emphasizes the molecular heterogeneity of DEB and provides more information for studies on genotype-phenotype correlations in different DEB subtypes.

Adolescent↗

PubMed1999

Redefinition of exon 7 in the COL1A1 gene of type I collagen by an intron 8 splice-donor-site mutation in a form of osteogenesis imperfecta: influence of intron splice order on outcome of splice-site mutation.

Most splice-site mutations lead to a limited array of products, including exon skipping, use of cryptic splice-acceptor or -donor sites, and intron inclusion. At the intron 8 splice-donor site of the COL1A1 gene, we identified a G+1-->A transition that resulted in the production of several splice products from the mutant allele. These included one in which the upstream exon 7 was extended by 96 nt, others in which either intron 8 or introns 7 and 8 were retained, one in which exon 8 was skipped, and one that used a cryptic donor site in exon 8. To determine the mechanism by which exon-7 redefinition might occur, we examined the order of intron removal in the region of the mutation by using intron/exon primer pairs to amplify regions of the precursor nuclear mRNA between exon 5 and exon 10. Removal of introns 5, 6, and 9 was rapid. Removal of intron 8 usually preceded removal of intron 7 in the normal gene, although, in a small proportion of copies, the order was reversed. The proportion of abnormal products suggested that exon 7 redefinition, intron 7 plus intron 8 inclusion, and exon 8 skipping all represented products of the impaired rapid pathway, whereas the intron-8 inclusion product resulted from use of the slow intron 7-first pathway. The very low-abundance cryptic exon 8 donor site product could have arisen from either pathway. These results suggest that there is commitment of the pre-mRNA to the two pathways, independent of the presence of the mutation, and that the order and rate of intron removal are important determinants of the outcome of splice-site mutations and may explain some unusual alterations.

Amino Acid Sequence↗

PubMed2005

Compound heterozygosity for new splice site mutations in the plakophilin 1 gene (PKP1) in a Chinese case of ectodermal dysplasia-skin fragility syndrome.

Ectodermal dysplasia-skin fragility syndrome is a rare autosomal recessive inherited disease characterized by skin fragility, nail dystrophy and hyperkeratosis of palms and soles. Skin biopsy shows the loss of cell adhesion and the decrease of desmosomes in number and size. Mutations in PKP1 have been found to be the underlying cause of the syndrome. We report here a Chinese case of ectodermal dysplasia-skin fragility syndrome. Mutation analysis revealed compound heterozygosity for mutations in PKP1 of the proband. A new splice site mutation (c.1053 T>A+c.1054+1 G>T) near the 3' end of exon 5 and at the donor end of intron 5 on one allele was transmitted from the proband's mother. Another new splice site mutation (c.1835-2 A>G) near the acceptor end of intron 10 originated from her father. The absence of the mutant mRNA and plakophilin 1 protein in the proband's skin may result from the mechanism of nonsense-mediated mRNA decay induced by premature stop codons in PKP1 transcripts due to the two splice site mutations.

Child, Preschool↗

PubMedDate not supplied

Identification of a novel splice site mutation in the human hairless gene underlying atrichia with papular lesions.

Atrichia with Papular Lesions (APL) is a rare autosomal recessive disorder characterized by complete hair loss that begins shortly after birth with the development of papular lesions on various regions of the body. Since the establishment of hairless (HR) gene mutations as the cause of this disorder, several patients previously assumed to suffer from alopecia universalis have been subsequently diagnosed with APL. In this study we have identified a novel splicing mutation, IVS8+2T-->G, in the hairless gene. This mutation most likely abolishes normal splicing of exon 8 and potentially leads to out-of-frame skipping of this exon and a downstream premature termination codon (PTC). Our findings contribute to the growing body of HR mutations implicated in APL and provide further evidence for the differentiation of APL from alopecia universalis.

Adult↗

PubMed1993

Further investigation of the HEXA gene intron 9 donor splice site mutation frequently found in non-Jewish Tay-Sachs disease patients from the British Isles.

In a previous study we found that a Tay-Sachs disease (TSD) causing mutation in the intron 9 donor splice site of the HEXA gene occurs at high frequency in non-Jewish patients and carriers from the British Isles. It was found more frequently in subjects of Irish, Scottish, and Welsh origin compared with English origin (63% and 31% respectively). We have now tested, in a blind study, 26 American TSD carriers and 28 non-carriers who have British ancestry for the intron 9 splice site mutation. Six of the carriers and none of the controls were positive for the mutation. All six had Irish ancestry, compared with nine of the 20 other (intron 9 mutation negative) TSD carriers (p < 0.05). These results confirm the previously found high frequency of the intron 9 mutation in non-Jewish TSD families of British Isles, particularly Irish, origin, and reinforce the need to screen such families for this mutation.

England↗

PubMed2003

A splice junction mutation in muscle carnitine palmitoyltransferase II deficiency.

We report the first splice junction mutation to be described in the carnitine palmitoyltransferase (CPT) 2 gene in a patient with the muscle form of CPT II deficiency. The patient, a 25-year-old man, suffered from attacks of myalgia and muscle weakness in early adult life. There was biochemical evidence of CPT II deficiency. Molecular genetic analysis revealed the common S113L mutation on one allele whilst a novel mutation at the splice donor junction in intron 3 was identified on the other allele. Sequencing of reverse transcription polymerase chain reaction (RT-PCR) products clearly demonstrated that this mutation causes the skipping of exon 3, thus establishing its pathogenic role.

Adult↗

PubMed1986

Mutations in a yeast intron demonstrate the importance of specific conserved nucleotides for the two stages of nuclear mRNA splicing.

Mutations were introduced at all positions of the internal conserved sequence (ICS) and at three positions in the 5' junction sequence of a Saccharomyces cerevisiae actin intron contained within an actin-thymidine kinase fusion gene. Stage I of splicing is reduced by changes at all these positions. C or A replacement at the fifth nucleotide of the 5' sequence reduces the fidelity of RNA cleavage at the 5' exon-intron junction and results in an accumulation of aberrant lariat intermediate. Stage II of splicing is affected by changes in the first and second residues of the 5' sequence and in the penultimate position of the ICS. An A to G transition at the branch point of the ICS causes a major accumulation of lariat intermediate.

Actins↗

PubMed1990

Analysis of a splice acceptor site mutation which produces multiple splicing abnormalities in the human argininosuccinate synthetase locus.

The cloned argininosuccinate synthetase gene from a citrullinemia patient's fibroblast cell line revealed a single base substitution (G to C) within the splice acceptor site of the last intron. The mutation abolished normal RNA splicing, and, by cDNA analysis, three abnormal splicing pathways were demonstrated. The major pathway involved the activation of a cryptic acceptor site in the last exon that resulted in a deletion of seven nucleotides in the mature RNA. Another pathway involved a downstream cryptic acceptor site, that is 388 nucleotides downstream from the first cryptic site. Northern blot analysis showed that this second cryptic site is present on the minor 2.7-kilobase mRNA, but not on the major species of argininosuccinate synthetase mRNA, which is 1.7-kilobases in length. Using this aberrant cDNA as a probe, the cDNA of the 2.7-kilobase mRNA was isolated and studied. Sequence analysis suggests that this species of RNA is the one that bypasses the polyadenylation signal employed by the 1.7-kilobase RNA. Since both transcripts encounter the same translation termination codon, both RNAs should encode identical protein. Furthermore, a tract of 22 repeats of d(CA).(GT) is found at the 3' end of the gene and this repeat sequence is present on the 2.7-kilobase RNA. The third pathway of the abnormal splicing revealed a rare class of transcript that has the last intron retained in the mature RNA. This study shows that in human the intron inclusion can occur through a naturally occurring point mutation. All these abnormally spliced RNAs resulted in a protein reading frame shift.

Amino Acid Metabolism, Inborn Errors↗

PubMed1998

Phenotypic variability associated with 14 splice-site mutations in the NF2 gene.

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder caused by mutations in the NF2 gene. Patients carrying NF2 mutations are predisposed to cerebral and spinal tumors with bilateral vestibular schwannomas as the hallmark. Using single strand conformation polymorphism and temperature gradient gel electrophoresis analysis, we have screened 87 unrelated NF2 patients for mutations in the NF2 gene. In this study, we report phenotypes associated with 14 splice-site mutations carried by 14 propositi and 11 relatives. The mutations were distributed in exons 2, 3, 5, 7, 8, 14, and 15. These splice-site mutations were associated with various phenotypes, from severe to asymptomatic. Phenotypic variation was also observed within families. Mutations downstream from exon 8 resulted more often in mild phenotypes. No meningiomas were found in any of 13 affected or mutation bearing individuals from three families with splice-site mutations of exons 14 and 15. These data suggest that splice-site alteration is a relatively common cause of NF2, and that unlike other mutations the clinical outcomes of splice-site mutations in the NF2 gene are variable. These results add to the growing body of information on genotype-phenotype correlation in NF2.

Adolescent↗