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Results for “Splicing mutations”

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PubMed1988

Mild hemophilia A associated with a cryptic donor splice site mutation in intron 4 of the factor VIII gene.

Hemophilia A, an X-linked disease caused by deficiency of factor VIII, is characterized by variation in clinical severity and coagulation activity. This variation is though to reflect heterogeneity of mutations in the factor VIII gene. Here we describe a CG-to-CA mutation within a potential cryptic donor splice site in intron 4 of the factor VIII gene from a patient with mild disease. This mutation makes the cryptic sequence resemble more closely the consensus sequence for donor splice sites. We infer that the mutation activates the cryptic donor splice site, which in turn causes a defect in RNA processing.

Adult↗
PubMed2005

Dominant optic atrophy caused by a novel OPA1 splice site mutation (IVS20+1G-->A) associated with intron retention.

Dominant optic atrophy (DOA) is the most common form of inherited primary optic neuropathy. The purpose of the current study was to report a novel OPA1 splice site mutation and investigate the impact of the mutation on pre-mRNA splicing in a female proband and her father diagnosed with DOA. We evaluated visual acuity, retinal fundi and kinetic visual fields. Color vision phenotypes were determined using the Farnsworth Panel D-15 and the Farnsworth-Munsell 100-hue tests. All 28 coding exons of the OPA1 gene were analyzed with polymerase chain reaction (PCR) amplification and direct sequencing. Total RNA extraction from white blood cells followed by reverse transcription-PCR (RT-PCR) was performed. We identified a novel heterozygous G to A mutation at position +1 of intron 20 (g.IVS20+1G-->A) in both patients. RT-PCR analysis revealed that the first 25 bp from intron 20 plus exon 20 were spliced onto exon 21. No difference in expression of mutant and wild-type transcripts was found within the linear range of amplification. Clinically, both patients exhibited reduced visual acuities, pallor of optic discs, decreased sensitivities of central visual fields and blue-yellow color vision defects. Previously, only one mechanism (skipping of exon) of pre-mRNA splicing defects has been reported among OPA1 splice site mutations. Our study demonstrates that the mechanism of intron retention is a novel type of pre-mRNA splicing defects. The mutant transcript with a premature termination codon is likely to encode a truncated protein, due to a translational frameshift (V672fsX675), that lacks 289 amino acids of the C-terminal end. Therefore, it is suggested that haploinsufficiency underlies DOA in the patients. However, we could not exclude the possibility that the truncated protein has a dominant negative activity because the mutant transcript is insusceptible to nonsense-mediated mRNA decay.

Adult↗
PubMed2006

A splice-site mutation in CCM1/KRIT1 is associated with retinal and cerebral cavernous hemangioma.

PURPOSE: To report a case of a unilateral retinal cavernous hemangioma associated with a novel splice-site mutation in CCM1/KRIT1. METHODS: An 11-year-old girl was noted to have an asymptomatic retinal cavernous hemangioma in the left eye. CCM1/KRIT1 was screened for mutations. RESULTS: Genetic evaluation of CCM1/KRIT1 revealed a single guanine-to-cytosine transversion in the invariant splice acceptor consensus sequence of intron 8 (c.1146-1G-->C), which is predicted to result in abnormal protein splicing. CONCLUSIONS: Mutations in CCM1/KRIT1 may be found in asymptomatic patients with retinal cavernous hemangioma.

Ankyrin Repeat↗
PubMed2003

Identification of a novel splice site mutation of CLCN5 gene and characterization of a new alternative 5' UTR end of ClC-5 mRNA in human renal tissue and leukocytes.

Mutations in the CLCN5 gene have been detected in Dent's disease and its phenotypic variants (X-linked recessive nephrolithiasis, X-linked recessive hypophosphatemic rickets, and idiopathic low-molecular-weight proteinuria of Japanese children). Dent's disease is a tubular disorder characterized by low-molecular-weight proteinuria, and nephrolithiasis associated with nephrocalcinosis and hypercalciuria. ClC-5 is the first chloride channel for which a definitive role in the trafficking and acidification-dependent recycling of apical membrane proteins has been established. In the course of CLCN5 SSCP analysis in patients with hypercalciuric nephrolithiasis, we detected a novel mutation at intron 2 of the CLCN5 gene, a T-to-G substitution, located 17 bp upstream of the AG acceptor site. To determine the effect of IVS2-17 T>G mutation on the correct splicing of intron 2, we studied ClC-5 transcripts in a patient's peripheral blood leukocytes by means of quantitative comparative RT/PCR, and found a new ClC-5 5' UTR isoform characterized by the untranslated exon 1b and by retention of intron 1b. This new isoform--isoform B1--was not correlated with mutation since it was detected also in control leukocytes and in renal tissues of kidney donors, thus confirming its physiological role. By RACE analysis we determined the putative transcriptional start site which is located at intron 1a, 251 nt upstream of the first nucleotide of the untranslated exon 1b. ORF analysis revealed that intron 1b retention in isoform B1 stabilizes the initiation of translation to the AGT at position 297 of the ClC-5 cDNA coding region.

5' Untranslated Regions↗
PubMed1995

Splice site mutation in the glucocorticoid receptor gene causes resistance to glucocorticoid-induced apoptosis in a human acute leukemic cell line.

Induction of apoptosis is the molecular basis for the therapeutic application of glucocorticoids (GC) in human leukemia. The beneficial effect of endocrine therapy is, however, hampered by the occurrence of resistant clones evolving under selective GC pressure. To delineate molecular mechanisms of GC resistance, we PCR amplified, cloned, and sequenced GC receptor (GR) transcripts and gene segments from a GC-resistant subclone of the human acute leukemic cell line CCRF-CEM, termed CEM-R6. Our analyses revealed that one GR gene allele harbored a point mutation (L753F) previously shown to compromise GR functions in other CCRF-CEM derivatives. On the second allele, we identified an A to G point mutation in the 3'-splice junction of intron G. As a consequence, a cryptic splice site 8 base pairs downstream within exon 8 is recognized, which leads to an 8-base deletion in the GR mRNA, resulting in reading frame shift and 2 consecutive in-frame preterminal stop codons. Translation of this mutant mRNA would produce a truncated GR protein missing 93 amino acids of the ligand-binding domain and expressing 9 altered residues at its new COOH terminus. In concert with the L753F mutation on the other allele, this molecular defect explains the GC-resistant phenotype and provides further evidence for mutational GR gene inactivation as a mechanism for human leukemic cells to escape GC-induced apoptosis.

Amino Acid Sequence↗
PubMed1999

Compound heterozygosity for novel splice site mutations in the BPAG2/COL17A1 gene underlies generalized atrophic benign epidermolysis bullosa.

Generalized atrophic benign epidermolysis bullosa, GABEB (OMIM# 226650), is a nonlethal variant of epidermolysis bullosa with autosomal recessive inheritance pattern. The pathogenesis of this disorder can be caused by mutations affecting two different gene/protein systems. Most of the mutations have been identified in the BPAG2/COL17A1 gene encoding a hemidesmosomal transmembrane protein, the 180 kDa bullous pemphigoid antigen (BP180), also known as type XVII collagen. The minority of the mutations are localized in the LAMB3 gene encoding the beta3 polypeptide of laminin 5. In In this study we describe a GABEB patient who showed absent expression of BP180 in the cultured keratinocytes as well as in the skin. The patient was a compound heterozygote for two different splice site mutations, 3053-1G-->C and 3871+1G-->C, affecting the extra-cellular domain of the protein. These mutations resulted in multiple aberrant splice variants, three of them causing premature termination codons for translation. This case, dealing with out-of-frame splice site mutations in BPAG2/COL17A1, attests to the molecular heterogeneity of GABEB.

Adult↗
PubMed1997

NF1 mutation analysis using a combined heteroduplex/SSCP approach.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized predominantly by neurofibromas, café-au-lait spots, and Lisch nodules. The disease is caused by disruptive mutations of the large NF1 gene, with half of cases caused by new mutation. Less than 100 constitutional mutations have thus far been published, ranging from very large deletions to point mutations. We have pursued NF1 mutation analysis by heteroduplex analysis (HDA) and single-strand conformational polymorphism analysis (SSCP) of individual exons. We streamlined these techniques to eliminate the use of radioactivity, to apply both methods to the same PCR product, and to multiplex samples in gels. Applied simultaneously to a set of 67 unrelated NF1 patients, HDA and SSCP have thus far identified 26 mutations and/or variants in 45 of the 59 exons tested. Disease-causing mutations were found in 19% (13/67) of cases studied. Both techniques detected a variety of mutations including splice mutations, insertions, deletions, and point changes, with some overlap in the ability of each method to detect variants.

Base Sequence↗
PubMed2000

Glucocerebrosidase gene mutations in patients with type 2 Gaucher disease.

Gaucher disease, the most common lysosomal storage disorder, results from the inherited deficiency of the enzyme glucocerebrosidase. Three clinical types are recognized: type 1, non-neuronopathic; type 2, acute neuronopathic; and type 3, subacute neuronopathic. Type 2 Gaucher disease, the rarest type, is progressive and fatal. We have performed molecular analyses of a cohort of 31 patients with type 2 Gaucher disease. The cases studied included fetuses presenting prenatally with hydrops fetalis, infants with the collodion baby phenotype, and infants diagnosed after several months of life. All 62 mutant glucocerebrosidase (GBA) alleles were identified. Thirty-three different mutant alleles were found, including point mutations, splice junction mutations, deletions, fusion alleles and recombinant alleles. Eleven novel mutations were identified in these patients: R131L, H255Q, R285H, S196P, H311R, c.330delA, V398F, F259L, c.533delC, Y304C and A190E. Mutation L444P was found on 25 patient alleles. Southern blots and direct sequencing demonstrated that mutation L444P occurred alone on 9 alleles, with E326K on one allele and as part of a recombinant allele on 15 alleles. There were no homozygotes for point mutation L444P. The recombinant alleles that included L444P resulted from either reciprocal recombination or gene conversion with the nearby glucocerebrosidase pseudogene, and seven different sites of recombination were identified. Homozygosity for a recombinant allele was associated with early lethality. We have also summarized the literature describing mutations associated with type 2 disease, and list 50 different mutations. This report constitutes the most comprehensive molecular study to date of type 2 Gaucher disease, and it demonstrates that there is significant phenotypic and genotypic heterogeneity among patients with type 2 Gaucher disease. Hum Mutat 15:181-188, 2000. Published 2000 Wiley-Liss, Inc.

Alleles↗
PubMed2000

Phenylketonuria and hyperphenylalaninemia in eastern Germany: a characteristic molecular profile and 15 novel mutations.

Phenylketonuria (PKU) is an important error of amino acid metabolism which results in most patients from phenylalanine hydroxylase (PAH) deficiency. PKU displays a marked genotypic heterogeneity both within and between different populations. The aim of this study was to establish the genotypic spectrum of PKU in eastern Germany, and to compare this to the distribution of mutations in western Germany. The study population included 302 patients in 290 families who were followed at treatment centers in Berlin, Leipzig and Jena. The study showed marked genotypic variability with a total of 75 mutations, including 15 that have so far not been described (eleven missense mutations, one splicing mutation, and three small deletions). One of these novel mutations, E183Q, occurred in cis to a R408W mutation. In the non-immigrant eastern German population, the frequency of R408W accounted for 40.1% of the PKU alleles. In the immigrant Turkish population of the former West Berlin, the most prevalent mutation was IVS10-11G>A (57%). There was a marked difference of the genotypic spectrum between the population studied here and the data reported from the western part of the country.

Amino Acid Substitution↗
PubMed2000

Genomic organization of the human phosphomannose isomerase (MPI) gene and mutation analysis in patients with congenital disorders of glycosylation type Ib (CDG-Ib).

CDG-Ib is the "gastro-intestinal" type of the congenital disorders of glycosylation (CDG) and a potentially treatable disorder. It has been described in patients presenting with congenital hepatic fibrosis and protein losing enteropathy. The symptoms result from hypoglycosylation of serum- and other glycoproteins. CDG-Ib is caused by a deficiency of mannose-6-phosphate isomerase (synonym: phosphomannose isomerase, EC 5.3.1.8), due to mutations in the MPI gene. We determined the genomic structure of the MPI gene in order to simplify mutation detection. The gene is composed of 8 exons and spans only 5 kb. Eight (7 novel) different mutations were found in seven patients with a confirmed phosphomannose isomerase deficiency, analyzed in the context of this study: six missense mutations, a splice mutation and one insertion. In the last, the mutation resulted in an unstable transcript, and was hardly detectable at the mRNA level. This emphasizes the importance of mutation analysis at the genomic DNA level.

Congenital Disorders of Glycosylation↗
PubMed2001

Molecular analysis of phenylketonuria (PKU) in newborns from Texas.

This study describes the mutations at the phenylalanine hydroxylase (PAH) locus in patients with the diagnosis of classic PKU (n=18), hyperphenylalaninemia (HPA) variant (n=9) and benign persistent hyperphenylalaninemia (HPA) (n=13) who were identified by the Texas Newborn Screening Program. Blinded studies were done by sequencing of the 13 exons and exon-intron boundaries of the PAH gene in genomic DNA isolated from dry blood spots. Thirty-six different mutations, including 25 missense mutations, six splice mutations, three deletions and two nonsense mutations were detected in 75 of the 80 mutant alleles (94%). The prevalent mutations were R408W (19%), V388M and IVS10nt-11g->a (6% each), Y414C (5%) and H170D, A403V, T380M and IVS7nt1g->a (4% each). Two novel missense mutations were identified in exon 5 (H170D and N167S). There was genotype/phenotype correlation in 33/40 cases (83%). For this population, exons 12, 11, 7, 5 and 8, which carry 78% of the mutations, would have to be screened first. However, the other exons must be studied when either one or no mutations are found in the primary screening. Hum Mutat 17:523, 2001.

Alleles↗
PubMed1992

Detection of a common mutation of the catalase gene in Japanese acatalasemic patients.

Acatalasemia was one of the earliest described genetic enzyme defects. In 1990, a causal point mutation (a splicing mutation) was first reported in a Japanese patient with acatalasemia. In the present study, the polymerase chain reaction and single-strand conformation polymorphism analysis were used to determine whether the same point mutation was present in unrelated Japanese patients. The subjects studied were the previously examined acatalasemic female, her brother, who is hypocatalasemic, and two other unrelated acatalasemic patients. A single G to A point mutation at the fifth position of intron 4, identical to that previously found, was present in all the studied patients. This finding strongly suggests that only a single mutated allele has spread in the Japanese population.

Acatalasia↗
PubMed2000

Missense mutations in SURF1 associated with deficient cytochrome c oxidase assembly in Leigh syndrome patients.

We have studied the fibroblasts of three patients suffering from Leigh syndrome associated with cytochrome c oxidase deficiency (LS-COX-). Their mitochondrial DNA was functional and all nuclear COX subunits had a normal sequence. The expression of transcripts encoding mitochondrial and nuclear COX subunits was normal or slightly increased. Similarly, the OXA1 transcript coding for a protein involved in COX assembly was increased. However, several COX-protein subunits were severely depressed, indicating deficient COX assembly. Surf1, a factor involved in COX biogenesis, was recently reported as mutated in LS-COX- patients, all mutations predicting a truncated protein. Sequence analysis of SURF1 gene in our three patients revealed seven heterozygous mutations, six of which were new : an insertion, a nonsense mutation, a splicing mutation of intron 7 in addition to three missense mutations. The mutation G385 A (Gly124-->Glu) changes a Gly that is strictly conserved in Surfl homologs of 12 species. The substitution G618 C (Asp202-->His), changing an Asp that is conserved only in mammals, appears to be a polymorphism. The mutation T751 C changes Ile246 to Thr, a position at which a hydrophobic amino acid is conserved in all eukaryotic and some bacterial species. Replacing Ile246 by Thr disrupts a predicted beta sheet structure present in all higher eukaryotes. COX activity could be restored in fibroblasts of the three patients by complementation with a retroviral vector containing normal SURF1 cDNA. These mutations identify domains essential to Surf1 protein structure and/or function.

Amino Acid Sequence↗