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Results for “Splicing mutations”

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PubMed2006

Novel splice site mutation in the caveolin-3 gene leading to autosomal recessive limb girdle muscular dystrophy.

Mutations in CAV3 gene encoding the protein caveolin-3 are associated with autosomal dominant limb girdle muscular dystrophy 1C, rippling muscle disease, hyperCKemia, distal myopathy, hypertrophic cardiomyopathy and rare autosomal recessive limb girdle muscular dystrophy phenotypes. In a 57-year-old patient with asymmetric limb girdle weakness, we detected a novel homozygous intronic mutation (IVS1 + 2T > C) of the CAV3 gene. This is the first splicing mutation reported for CAV3. These findings add to the clinical and genetic variability of CAV3 mutations.

Base Sequence↗

PubMed2002

Acanthocytosis in a patient with homozygous familial hypobetalipoproteinemia due to a novel APOB splice site mutation.

We report on acanthocytosis in a 31-year-old woman with homozygous familial hypobetalipoproteinemia due to a mutation affecting the splicing of the APOB gene encoding apolipoprotein B. Treatment with fat-soluble vitamins was associated with arrest of the usually progressive neurological complications of this condition. However, the acanthocytosis - literally 'thorny' erythrocytes that result from abnormal membrane fluidity - persists despite treatment.

Acanthocytes↗

PubMed1998

Cycloheximide facilitates the identification of aberrant transcripts resulting from a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa.

Patients with generalized atrophic benign epidermolysis bullosa often show decreased expression of type XVII collagen, a transmembrane hemidesmosomal protein encoded by COL17A1. This report documents a novel splice-site mutation in COL17A1 in a patient with generalized atrophic benign epidermolysis bullosa, and applies a new methodology to define and characterize the resulting mRNA splice variants. Mutational analysis of COL17A1 identified a maternally inherited G-to-T transversion at the -1 position of exon 32. This acceptor splice-site mutation led to the formation of aberrant transcripts present at extremely low levels. Based on our recent finding that cycloheximide stabilized mutant COL17A1 transcripts in keratinocytes homozygous for a frameshift mutation, the effects of the splice-site mutation on splicing of COL17A1 transcripts were determined using reverse transcriptase polymerase chain reaction of total RNA from keratinocytes incubated for 2.5 h in the presence or absence of 10 microg cycloheximide per ml. Using this approach, an abnormally spliced transcript was identified that contains an extra 264 bases upstream from exon 32, resulting in a premature termination codon 27 bp downstream from the cryptic splice site. Three other splice variants, including one derived from the skipping of exon 32, were also identified. These results indicate the usefulness of cycloheximide treatment in evaluating the abnormal processing of mRNA due to splice-site mutations, because: (i) aberrant splicing often generates a premature termination codon, (ii) transcripts with premature termination codons can occur at low or undetectable levels due to nonsense-mediated mRNA decay, and (iii) the levels of these transcripts can be increased by cycloheximide.

Adult↗

PubMed1997

Two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency.

Genetic analysis revealed two distinct novel splice site mutations in a compound heterozygous patient with protein S deficiency. The paternal mutation was a G-to-T transition at position-1 of the acceptor splice site of intron N (Mutation I), and the maternal mutation was a G-to-C transversion at position-1 of the donor splice site of intron C (Mutation II). Both splice site mutations decreased the mutated mRNA accumulation to the same extent, approximately 40% of the normal mRNA. However, the mutations were associated with different phenotypical expressions: the paternal mutant protein S was not detected in vivo, while the maternal mutant protein S was present in the plasma in reduced quantity. Because Mutation I caused a cryptic splicing in the mutated mRNA, resulting in a reading frameshift and premature termination, the predicted mutant protein S might be highly unstable. In contrast. Mutation II led to the substitution of Va146 by Leu, which might be much less deleterious for the synthesis, secretion and stability of the predicted mutant protein S. It was supposed that the different post-translational metabolisms produced the distinct phenotypical expressions of the mutations.

Adult↗

PubMed2002

Pyruvate dehydrogenase deficiency as a result of splice-site mutations in the PDX1 gene.

Mutations in the E3-binding protein component of pyruvate dehydrogenase complex have been demonstrated in a few cases of Leigh syndrome. We report that two mutations previously detected in the E3-binding protein cDNA are the consequence of splice-site mutations. Both involved a single base substitution in the conserved dinucleotides of splice junctions, one leading to skipping of an exon and the other, to activation of a cryptic site. Our findings add to the understanding of molecular basis of E3-binding protein deficiency and indicate yet again the high frequency of splicing mutations in this gene.

Amino Acid Sequence↗

PubMed1994

Correct splicing despite mutation of the invariant first nucleotide of a 5' splice site: a possible basis for disparate clinical phenotypes in siblings with adenosine deaminase deficiency.

Adenosine deaminase (ADA) deficiency usually causes severe combined immune deficiency in infancy. Milder phenotypes, with delayed or late onset and gradual decline in immune function, also occur and are associated with less severely impaired deoxyadenosine (dAdo) catabolism. We have characterized the mutations responsible for ADA deficiency in siblings with striking disparity in clinical phenotype. Erythrocyte dAdo nucleotide pool size, which reflects total residual ADA activity, was lower in the older, more mildly affected sib (RG) than in her younger, more severely affected sister (EG). Cultured T cells, fibroblasts, and B lymphoblasts of RG had detectable residual ADA activity, while cells of EG did not. ADA mRNA was undetectable by northern analysis in these cells of both patients. Both sibs were found to be compound heterozygotes for the following novel splicing defects: (1) a G+1-->A substitution at the 5' splice site of IVS 2 and (2) a complex 17-bp rearrangement of the 3' splice site of IVS 8, which inserted a run of seven purines into the polypyrimidine tract and altered the reading frame of exon 9. PCR-amplified ADA cDNA clones with premature translation stop codons arising from aberrant pre-mRNA splicing were identified, which were consistent with these mutations. However, some cDNA clones from T cells of both patients and from fibroblasts and Epstein-Barr virus (EBV)-transformed B cells of RG, were normally spliced at both the exon 2/3 and exon 8/9 junctions. A normal coding sequence was documented for clones from both sibs. The normal cDNA clones did not appear to arise from either contamination or PCR artifact, and mosaicism seems unlikely to have been involved. These findings suggest (1) that a low level of normal pre-mRNA splicing may occur despite mutation of the invariant first nucleotide of the 5' splice donor sequence and (2) that differences in efficiency of such splicing may account for the difference in residual ADA activity, immune dysfunction, and clinical severity in these siblings.

Adenosine Deaminase↗

PubMed1991

Occurrence of multiple aberrantly spliced mRNAs upon a donor splice site mutation that causes familial lipoprotein lipase deficiency.

A donor splice site mutation was found in the lipoprotein lipase (LPL) gene of a patient with familial LPL deficiency. The mutation, a G----A substitution, occurred at the first nucleotide of intron 2. Northern blot analysis of total RNA from the patient showed strikingly low levels of LPL-specific mRNAs. Using the polymerase chain reaction, the LPL mRNA splicing was analyzed in detail. The results demonstrated that no normal splicing occurred at the authentic splice site; rather a cryptic splice site 18 bases upstream from the mutation site was preferentially utilized. Although the resulting alteration in mRNA was a minute in-frame 18-base deletion, the amount of the abnormal transcript was only 1/12 that of the normal. In addition to this major cryptic splice site, we also identified multiple minor sites which were utilized at extremely lower efficiencies. Unexpectedly, one of these minor sites was also used as an alternative splice site in the normal subject at a comparably low efficiency. The sequences of these minor cryptic sites possessed many of the characteristics common to those of other normal splice sites, indicating that even such minor sites should have also been selected according to the general rules for splice site selection. These results demonstrate that upon mutation, a broad spectrum of cryptic splice sites is activated in vivo at the sites' respective efficiencies.

Adult↗

PubMed2006

NF1 mutation rather than individual genetic variability is the main determinant of the NF1-transcriptional profile of mutations affecting splicing.

A significant number of neurofibromatosis type 1 (NF1) mutations result in exon skipping. The majority of these mutations do not occur in the canonical splice sites and can produce different aberrant transcripts whose proportions have not been well studied. It has been hypothesized that differences in the mutation-determined NF1-transcriptional profile could partially explain disease variability among patients bearing the same NF1 splice defect. In order to gain insight into these aspects, we analyzed the proportion of the different transcripts generated by nine NF1-splicing mutations in 30 patients. We assessed the influence of the mutation in the NF1-related transcriptional profiles and investigated the existence of individual differences in a global manner. We analyzed potential differences in tissue-specific transcriptional profiles and evaluated the influence of sample processing and mRNA nonsense-mediated decay (NMD). Small transcriptional differences were found in neurofibromas and neurofibroma-derived Schwann cells (SC) compared to blood. We also detected a higher cell culture-dependent NMD. We observed that mutation per se explains 93.5% of the profile variability among mutations studied. However, despite the importance of mutation in determining the proportion of NF1 transcripts generated, we found certain variability among patients with the same mutation. From our results, it seems that genetic factors influencing RNA processing play a minor role in determining the NF1-transcriptional profile. Nevertheless neurofibromin studies would clarify whether these small differences translate into significant functional changes that could explain the great clinical expressivity observed in the disease or any of the disease-related traits.

Alleles↗

PubMed2001

A novel 3'-splice site mutation in peripheral myelin protein 22 causing hereditary neuropathy with liability to pressure palsies.

Hereditary neuropathy with liability to pressure palsies (HNPP) is an autosomal dominant, demyelinating peripheral neuropathy. Clinical hallmarks are recurrent painless focal neuropathies mostly preceded by minor trauma or compression at entrapment sites of peripheral nerves. In the majority of the patients, HNPP is caused by a 1.5 Mb deletion on chromosome 17p11.2-p12 containing the peripheral myelin protein 22 (PMP22) gene. Point mutations within this gene are reported in only a few families. We report a novel mutation in the PMP22 gene in a Spanish family with HNPP. The mutation is a 3' splice-site mutation, preceding coding exon 3 (c.179-1 G>C), causing a mild HNPP phenotype.

Adult↗

PubMed2000

Splice-site mutation in TGM1 in congenital recessive ichthyosis in American families: molecular, genetic, genealogic, and clinical studies.

Lamellar ichthyosis (LI, OMIM no. 242300) is a severe autosomal recessive genodermatosis with an estimated prevalence of 1:200,000. LI represents one end of the spectrum of congenital recessive ichthyosis (CRI). Mutations in the gene for transglutaminase-1 (TGM1) are responsible for many cases of LI and occur throughout the coding sequence of the gene. Our analyses of patients with CRI revealed a common TGM1 mutation involving loss of the intron 5 splice acceptor site leading to alternative splicing of the message. We found families in which the splice acceptor site mutation was homozygous, and families where the patients were compound heterozygotes for the splice acceptor site mutation and another TGM1 mutation. A mutation at this same site occurs in the majority of Norwegian patients as a founder effect. In our ethnically diverse patient population, none of whom have known Norwegian ancestry, haplotype analysis of the TGM1 chromosomal region also suggested the existence of a founder effect. Comparison of the common haplotype in our data with the Norwegian data showed that 2/7 of our splice acceptor site mutation chromosomes had the full reported Norwegian haplotype, and the remaining five chromosomes exhibited recombination at the most distal marker studied. History, family origins, and haplotype analysis suggested that the mutation originally arose on a German background and was introduced into Norway around 800-1000 AD. We also found a limited correlation between genotype and phenotype in our study, with the four homozygous patients having less severe disease than many of the heterozygotes, and no patient with a splice acceptor site mutation having erythroderma or a congenital ichthyosiform erythroderma phenotype.

Adolescent↗

PubMed1998

Growth hormone (GH) insensitivity syndrome with high serum GH-binding protein levels caused by a heterozygous splice site mutation of the GH receptor gene producing a lack of intracellular domain.

Most of the GH receptor (GHR) gene abnormalities causing GH insensitivity syndrome (GHIS) are located in the region coding the extracellular domain, and serum GH-binding protein (GHBP) levels, determined by ligand-mediated immunofunctional assay, are low in most of the patients with GHIS. We present here a heterozygous point mutation of the donor splice site in intron 9 of the GHR gene in two Japanese siblings with GHIS, whose serum GHBP levels were high. The same mutation was found in their mother as well. The analysis of ribonucleic acid from the peripheral leukocytes revealed complete skipping of exon 9 from one allele, but not the other, in the GHR complementary DNA and appearance of a premature stop codon in exon 10. The translated protein was truncated with deletion of 98% of the intracellular domain of the GHR, including boxes 1 and 2, which are critical for GH signal transduction and GHR internalization, respectively. Recently, it was shown that the truncated GHR lacking the intracellular domain was physiologically present in a minute amount, served as a negative regulator for GH signaling, and possessed increased capacity to generate GHBP. Therefore, the mutation found in our patients caused the pathogenetic production of the truncated GHR with a dominant negative effect on GH signaling, which is probably responsible for their short stature and high serum GHBP levels.

Adolescent↗

PubMed2001

[Splice site mutations and atherosclerosis: mechanisms and prediction models].

Nucleotide variants in genes of the lipid metabolism influence the risk of premature atherosclerosis. Ten percent of all single nucleotide substitutions in these genes involve splice sites. The effects of these changes on mRNA splicing and phenotypic severity, however, are not inherently obvious from the nucleotide sequence. This review presents various genes of lipid metabolism with splicing mutations known to influence the risk of premature atherosclerosis. Mechanisms of pre-mRNA splicing are illustrated and different models for prediction of the effect of nucleotide substitutions on splice-site function are presented. The role of information theory-based models is emphasized along with its role for prediction of splice-site function and phenotypic severity of atherosclerosis.

Age Factors↗

PubMed1998

Autosomal recessive retinitis pigmentosa and cone-rod dystrophy caused by splice site mutations in the Stargardt's disease gene ABCR.

Ophthalmological and molecular genetic studies were performed in a consanguineous family with individuals showing either retinitis pigmentosa (RP) or cone-rod dystrophy (CRD). Assuming pseudodominant (recessive) inheritance of allelic defects, linkage analysis positioned the causal gene at 1p21-p13 (lod score 4.22), a genomic segment known to harbor the ABCR gene involved in Stargardt's disease (STGD) and age-related macular degeneration (AMD). We completed the exon-intron structure of the ABCR gene and detected a severe homozygous 5[prime] splice site mutation, IVS30+1G->T, in the four RP patients. The five CRD patients in this family are compound heterozygotes for the IVS30+1G->T mutation and a 5[prime] splice site mutation in intron 40 (IVS40+5G->A). Both splice site mutations were found heterozygously in two unrelated STGD patients, but not in 100 control individuals. In these patients the second mutation was either a missense mutation or unknown. Since thus far no STGD patients have been reported to carry two ABCR null alleles and taking into account that the RP phenotype is more severe than the STGD phenotype, we hypothesize that the intron 30 splice site mutation represents a true null allele. Since the intron 30 mutation is found heterozygously in the CRD patients, the IVS40+5G->A mutation probably renders the exon 40 5[prime] splice site partially functional. These results show that mutations in the ABCR gene not only result in STGD and AMD, but can also cause autosomal recessive RP and CRD. Since the heterozygote frequency for ABCR mutations is estimated at 0.02, mutations in ABCR might be an important cause of autosomal recessive and sporadic forms of RP and CRD.

ATP-Binding Cassette Transporters↗

PubMed2002

An exon splice enhancer mutation causes autosomal dominant GH deficiency.

Familial isolated GH deficiency type II (IGHD II) is caused, in some cases, by heterogeneous IVS3 mutations that affect GH mRNA splicing. We report here our finding an A-->G transition of the fifth base of exon 3 (E3+ 5 A-->G) in affected individuals from an IGHD II family. This mutation disrupts a (GAA)(n) exon splice enhancer (ESE) motif immediately following the weak IVS2 3' splice site. The mutation also destroys an MboII site used to demonstrate heterozygosity in all affected family members. To determine the effect of ESE mutations on GH mRNA processing, GH(3) cells were transfected with expression constructs containing the normal ESE, +5 A-->G, or other ESE mutations, and cDNAs derived from the resulting GH mRNAs were sequenced. All ESE mutations studied reduced activation of the IVS2 3' splice site and caused either partial E3 skipping, due to activation of an E3+ 45 cryptic 3' splice site, or complete E3 skipping. Partial or complete E3 skipping led to loss of the codons for amino acids 32-46 or 32-71, respectively, of the mature GH protein. Our data indicate that the E3+ 5 A-->G mutation causes IGHD II because it perturbs an ESE required for GH splicing.

Adult↗

PubMed2005

The location of constitutional neurofibromatosis 2 (NF2) splice site mutations is associated with the severity of NF2.

Neurofibromatosis 2 (NF2) patients with constitutional splice site NF2 mutations have greater variability in disease severity than NF2 patients with other types of mutations; the cause of this variability is unknown. We evaluated genotype-phenotype correlations, with particular focus on the location of splice site mutations, using mutation and clinical information on 831 patients from 528 NF2 families with identified constitutional NF2 mutations. The clinical characteristics examined were age at onset of symptoms of NF2 and number of intracranial meningiomas, which are the primary indices of the severity of NF2. Two regression models were used to analyse genotype-phenotype correlations. People with splice site mutations in exons 1-5 had more severe disease than those with splice site mutations in exons 11-15. This result is compatible with studies showing that exons 2 and 3 are required for self-association of the amino terminal of the NF2 protein in vitro, and that deletions of exons 2 and 3 in transgenic and knockout mouse models of NF2 cause a high prevalence of Schwann cell derived tumours.

Alternative Splicing↗

PubMed2003

A novel splice site mutation in neonatal carnitine palmitoyl transferase II deficiency.

Mitochondrial beta-oxidation of long-chain fatty acids requires the concerted action of three tightly integrated membrane-bound enzymes (carnitine palmitoyltransferase I and II and carnitine/acylcarnitine translocase) that transport them into mitochondria. Neonatal onset of carnitine palmitoyltransferase II (CPT II) deficiency is an autosomal recessive, often lethal disorder of this transport. We describe a novel splice-site mutation in the CPT II gene, found in a Moroccan family, of which four out of five children have died from the neonatal form of CPT II deficiency. Mutation detection studies at the mRNA level in the CPT II gene implied that the affected children were homozygous for the previously reported 534T insertion followed by a 25-bp deletion (encompassing bases 534-558). Studies of genomic DNA, however, revealed all patients to be compound heterozygous for this 534T ins/del 25 mutation, and for a new g-->a splice-site mutation in the splice-acceptor site of intron 2. Because of these findings, prenatal diagnosis was performed in chorionic villi of three new pregnancies. This did not reveal new compound heterozygous genotypes, and, after uneventful pregnancies, all children appeared to be healthy. The new mutation is the first splice-site mutation ever identified in CPT II deficiency. The fact that it was not discovered in the patient's cDNA makes this study another example of the incompleteness of mutation detection at the mRNA level in cases where a mutation leads to aberrant splicing or nonsense-mediated messenger decay.

Carnitine O-Palmitoyltransferase↗

PubMed1993

A 5' splice junction mutation leading to exon deletion in an Ashkenazic Jewish family with phosphofructokinase deficiency (Tarui disease).

A deficiency of the muscle isoform of the enzyme, phosphofructokinase (PFK, EC 2.7.1.11), leads to an illness (glycogenosis, Type VII) characterized by myopathy and hemolysis. A patient with this disease and an affected sister were found to have a G to A substitution at the 5' donor site of intron 5 of the PFK-M gene. This mutation led to a splicing defect: a complete deletion of the preceding exon in the patient's mRNA. The patient, an affected sister, and related and unrelated family members, who were of Ashkenazic Jewish background, were screened for the mutation by denaturing gradient gel electrophoresis and by allele specific hybridization of genomic DNA. The affected sisters are homozygous for the mutation, and their children, who are unaffected, are heterozygous. The only previously characterized genetic defect in this disease, found in a Japanese patient, was a G to T mutation at the beginning of intron 15 with splicing to a cryptic site within exon 15 (1). Both mutations lead to inframe deletions, but of different parts of the protein. The differences between the two aberrant proteins may account for clinical differences between our patients and the Japanese patient.

Base Sequence↗

PubMed1992

Characterization of an intron 12 splice donor mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.

Cystic fibrosis, the most common lethal genetic disease in the white population, is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Analysis of DNA from a pancreatic insufficient patient by chemical mismatch cleavage and subsequent DNA sequencing led to the identification of a potential splice mutation in the CFTR gene. A transition of the invariant guanosine to adenosine (1898 + 1G > A) was found at the splice donor site of intron 12. To determine the effect of this mutation on the patient's CFTR transcripts, RNA from the nasal epithelium was reverse transcribed and amplified by the polymerase chain reaction (RT-PCR). Direct sequencing of the PCR products revealed that the transcript from the chromosome with the 1898 + 1G > A mutation had skipped exon 12 entirely, resulting in a joining of exons 11 and 13. Deletion of exon 12 results in the removal of a highly conserved region which encodes the Walker B consensus sequence of the first nucleotide-binding fold of CFTR.

Adult↗