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Results for “Splicing mutations”

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At least 73 records · Page 4Linked to original sources

PubMed2005

A novel splice mutation of HERG in a Chinese family with long QT syndrome.

Congenital long QT syndrome (LQTS) is a genetically heterogeneous disease in which six ion-channel genes have been identified. The phenotype-genotype relationships of the HERG (human ether-a-go-go-related gene) mutations are not fully understood. The objective of this study is to identify the underlying genetic basis of a Chinese family with LQTS and to characterize the clinical manifestations properties of the mutation. Single strand conformation polymorphism (SSCP) analyses were conducted on DNA fragments amplified by polymerase chain reaction from five LQT-related genes. Aberrant conformers were analyzed by DNA sequencing. A novel splice mutation in C-terminus of HERG was identified in this Chinese LQTS family, leading to the deletion of 11-bp at the acceptor splice site of Exon9 [Exon9 IVS del (-12-->-2)]. The mutation might affect, through deficient splicing, the putative cyclic nucleotide binding domain (CNBD) of the HERG K(+) channel. This mutation resulted in a mildly affected phenotype. Only the proband had a history of syncopes, while the other three individuals with long QT interval had no symptoms. Two other mutation carriers displayed normal phenotype. No sudden death occurred in the family. The 4 affected individuals and the two silent mutation carriers were all heterozygous for the mutation. It is the first splice mutation of HERG reported in Chinese LQTS families. Clinical data suggest that the CNBD mutation may be less malignant than mutations occurring in the pore region and be partially dominant over wild-type function.

Asian People↗

PubMed1997

Spectrin St Claude, a splicing mutation of the human alpha-spectrin gene associated with severe poikilocytic anemia.

An alpha-spectrin variant with increased susceptibility to tryptic digestion, alpha(II/47), was previously observed in a child with severe, recessively inherited, poikilocytic anemia. The molecular basis of this variant, spectrin St Claude, has now been identified as a splicing mutation of the alpha-spectrin gene due to a T --> G mutation in the 3' acceptor splice site of exon 20. This polypyrimidine tract mutation creates a new acceptor splice site, AT --> AG, and leads to the production of two novel mRNAs. One mRNA contains a 12 intronic nucleotide insertion upstream of exon 20. This insertion introduces a termination codon into the reading frame and is predicted to encode a truncated protein (108 kD) that lacks the nucleation site and thus cannot be assembled in the membrane. In the other mRNA, there is in-frame skipping of exon 20, predicting a truncated (277 kD) alpha-spectrin chain. The homozygous propositus has only truncated 277 kD alpha-spectrin chains in his erythrocyte membranes. His heterozygous parents are clinically and biochemically normal. This allele was identified in 3% of asymptomatic individuals from Benin, Africa.

Adult↗

PubMed2006

Functional analysis of splicing mutations and of an exon 2 polymorphic variant of SERPING1/C1NH.

Several sequence changes have been reported in hereditary angioedema patients in intron 2 of the SERPING1/C1NH gene, but their consequences on splicing have not been determined. We examined in cell transfection assays the consequences at the mRNA level of splicing mutations affecting either the +3 or the +5 donor site positions, or the conserved canonical splicing signals of exon 2, using mutant C1 inhibitor minigene constructs. Both intron 2 mutations, c.51+3A>G and c.51+5G>A, resulted in marked exon 2 skipping in these assays, but also yielded a large fraction of normal transcripts. We show that the c.51+3A>G mutation cosegregates with low C1 inhibitor protein levels in one family. Moreover, the second base of exon 2 of the SERPING1/C1NH gene is the site of a polymorphic variant, which has been proposed as a modifier of disease severity. We found that the c.-21C allele at this position yields low but significant levels of exon 2 skipping in transfected Hep G2 or Hep 3B cells, suggesting that this allele may contribute, at the RNA level, to more severe forms of angioedema. Furthermore, we describe a previously not detected alternative splicing of exon 3, found in peripheral blood cell mRNA but not in the liver or in hepatoma cell lines and we show that, in cultured monocytes of a patient carrying the c.51+3A>G mutation, this alternative splicing is shifted from exon 3 exclusion to skipping of both exons 2 and 3. The latter finding suggests that mutations affecting splicing of exon 2 of the SERPING1/C1NH gene may have different consequences in monocytes versus other cell types.

Adolescent↗

PubMed2006

GH-1 gene splicing mutations: molecular basis of hereditary isolated growth hormone deficiency in children.

Children, residents of the Russian Federation, with congenital isolated growth hormone deficiency, were screened for mutations of GH-1 gene, the main gene of this deficiency. Twenty-eight children from 26 families with total congenital isolated growth hormone deficiency were examined. Direct sequencing of GH-1 detected five splicing mutations in intron 2, intron 3, and exon 4, two of them were never described previously. Three dominant negative mutations of GH-1 splicing, the basis for autosomal dominant isolated growth hormone deficiency (type II), are presented: IVS2 -2A>T, IVS3 +2T>C, and IVS3 +1G A mutation can be regarded as the most incident in type II isolated growth hormone deficiency in the Russian population.

Base Sequence↗

PubMed1993

A novel germline p53 splicing mutation in a pediatric patient with a second malignant neoplasm.

A novel germline p53 splicing mutation was identified in a pediatric patient with two metachronous primary cancers that are constituent tumors of the Li-Fraumeni syndrome. Genomic DNA from the second tumor showed the same mutation and loss of heterozygosity at the p53 locus. The mutant mRNA and protein were present in the tumor tissue. In contrast, in the normal tissues bearing the germline mutation in the heterozygous state, predominantly normal mRNA was expressed and the mutant p53 protein was not detectable. The functional silence and relative lack of mutant p53 mRNA expression in the normal tissues of this patient may be caused by decreased stability or decreased production. If this proves a more general pattern of expression of mutant p53 in individuals with germline mutations, these findings may explain the paucity of tumors in individuals affected with the Li-Fraumeni syndrome.

Adult↗

PubMed1999

Splicing mutations in KCNQ1: a mutation hot spot at codon 344 that produces in frame transcripts.

BACKGROUND: Long-QT syndrome is a monogenic disorder that produces cardiac arrhythmias and can lead to sudden death. At least 5 loci and 4 known genes exist in which mutations have been shown to be responsible for the disease. The potassium channel gene KCNQ1, previously named KVLQT1, on chromosome 11p15.5 is one of these. METHODS AND RESULTS: We initially analyzed one family using microsatellite markers and found linkage to KCNQ1. Mutation detection showed a G to C change in the last base of exon 6 (1032 G-->C) that does not alter the coded alanine. Restriction digest analysis in the family showed that only affected individuals carried the mutation. A previous report suggested that a G to A substitution at the same position may act as a splice mutation in KCNQ1, but no data was given to support this hypothesis nor was the transcription product identified. We have shown by reverse-transcription polymerase chain reaction that 2 smaller bands were produced for the KCNQ1 gene transcripts in addition to the normal-sized transcripts when lymphocytes of affected individuals were analyzed. Sequencing these transcripts showed a loss of exon 7 in one and exons 6 and 7 in the other, but an in-frame transcript was left in each instance. We examined other families in whom long-QT syndrome was diagnosed and found another unreported splice-site mutation, 922-1 G-->C, in the acceptor site of intron 5, and 2 of the previously reported 1032 G-->A mutations. All these showed a loss of exons 6 and 7 in the mutant transcripts, validating the proposal that a consensus sequence is affected in the exonic mutations and that the integrity of the base at position 1032 is essential for correct processing of the transcript. CONCLUSIONS: The 6 cases already reported in the literature with the 1032 G-->A transition, the novel 1032 G-->C transversion, and a recent G-->T transversion at the same base show that codon 344 is the second most frequently mutated after codon 341, suggesting at least two hotspots for mutations in KCNQ1.

Child, Preschool↗

PubMed2002

Normal volume of distribution of tobramycin in a mother and daughter with a CFTR splice mutation (1717 - 1G --> A).

A mother and daughter pair with CF who shared a splice mutation (1717 - 1G --> A) had a normal volume of distribution of tobramycin. The literature on tobramycin pharmacokinetics, which was published before the genetic defect was identified, is discussed. The authors speculate on the role of CFTR in the distribution of aminoglycosides and recommend that CFTR mutations should be clarified in all future studies of tobramycin pharmacokinetics in patients with CF.

Adult↗

PubMed1995

An RNA-splicing mutation (G+5IVS20) in the type II collagen gene (COL2A1) in a family with spondyloepiphyseal dysplasia congenita.

Defects in type II collagen have been demonstrated in a phenotypic continuum of chondrodysplasias that includes achondrogenesis II, hypochondrogenesis, spondyloepiphyseal dysplasia congenita (SEDC), Kniest dysplasia, and Stickler syndrome. We have determined that cartilage from a terminated fetus with an inherited form of SEDC contained both normal alpha 1(II) collagen chains and chains that lacked amino acids 256-273 of the triple-helical domain. PCR amplification of this region of COL2A1, from genomic DNA, yielded products of normal size, while amplification of cDNA yielded a normal sized species and a shorter fragment missing exon 20. Sequence analysis of genomic DNA from the fetus revealed a G-->T transversion at position +5 of intron 20; the affected father was also heterozygous for the mutation. Allele-specific PCR and heteroduplex analysis of a VNTR in COL2A1 independently confirmed the unaffected status of a fetus in a subsequent pregnancy. Thermodynamic calculations suggest that the mutation prevents normal splicing of exon 20 by interfering with binding of U1 small-nuclear RNA to pre-mRNA, thus leading to skipping of exon 20 in transcripts from the mutant allele. Electron micrographs of diseased cartilage showed intracellular inclusion bodies, which were stained by an antibody to alpha 1(II) procollagen. Our findings support the hypothesis that alpha-chain length alterations that preserve the Gly-X-Y repeat motif of the triple helix result in partial intracellular retention of alpha 1(II) procollagen and produce mild to moderate chondrodysplasia phenotypes.

Adult↗

PubMed1990

Splicing mutations in the CHO DHFR gene preferentially induced by (+/-)-3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene.

Cultured Chinese hamster ovary (CHO) cells were treated with the polycyclic aromatic hydrocarbon racemic 3 alpha,4 beta-dihydroxy-1 alpha,2 alpha-epoxy-1,2,3,4- tetrahydrobenzo[c]phenanthrene. Mutants deficient in dihydrofolate reductase activity were isolated. A carcinogen treatment at 0.1 microM yielded at 46% survival of the treated population and an induced frequency of mutation of 1.7 x 10(-4), 10(3)-fold greater than the spontaneous rate. By polymerase chain reaction amplification and direct DNA sequencing, we determined the base changes in 38 mutants. Base substitutions accounted for 78% (30/38) of the mutations. We obtained, in addition, four frameshift and four complex mutations. The preferred type of mutation was transversion (A.T----T.A and G.C----T.A) occurring in 69% of the analyzed mutants. A purine was on the 3' side of the putative adduct site in every mutant. Mutations were favored at sequences AGG, CAG, and AAG (the underlined base is the target). Surprisingly, 42% of the mutations created mRNA splicing defects (16/38), especially at splice acceptor sites for each of the five introns. Thus, this chemical carcinogen may recognize some aspect of DNA structure in regions corresponding to pre-mRNA splice sites.

Animals↗

PubMed2001

Prenatal diagnosis of ATR-X syndrome in a fetus with a new G>T splicing mutation in the XNP/ATR-X gene.

The molecular cause of the alpha-thalassemia/mental retardation syndrome (ATR-X) resides in mutations affecting the XNP/ATR-X gene. Recently molecular defects in the gene have been found in singular cases of a discrete number of X-linked mental retardation (XLMR). ATR-X-affected males are characterised by severe mental retardation, distinct facial dysmorphisms and genital abnormalities, besides a wide spectrum of pathological features and an extremely limited biological fitness. Given that molecular investigation of XNP/ATR-X mutations is made onerous by the length of the gene transcript, we carried out a prenatal diagnosis in a fetus at risk for ATR-X syndrome by initially determining the XNP/ATR-X gene haplotype before considering gene sequencing. Disease-associated haplotype analysis was performed selecting five genic (CA)n repeats that showed high heterozygosity (Het>0.7) in the general population. The fetus segregated an identical allelic pattern to that of the affected child of the family under investigation who shows features suggestive of the ATR-X syndrome. Subsequent mutational analysis of the gene revealed a novel IVS3+1G>T splicing mutation confirming the diagnosis.

Adult↗

PubMed1990

Molecular analysis of human acatalasemia. Identification of a splicing mutation.

To search for the molecular defect of Japanese-type acatalasemia, we cloned the mutant catalase gene from a person with this deficiency. The nucleotide sequence of the mutant gene was determined for all exons, exon/intron junctions, and 5' and 3' flanking regions, and the findings were compared with the sequence from the normal gene. Seven base differences were found between the two genes. Among them, a G to A substitution at the fifth position of intron 4 (a splicing mutation) seemed most likely to be responsible for the defective catalase synthesis in the subject. To obtain suggestive evidence, we constructed chimeric genes that contained a segment of either the normal or mutant catalase gene, encompassing a 3' part of exon 4, the entire intron 4 and a 5' portion of exon 5, within the third exon of the human alpha-globin gene. When this chimeric gene construct was introduced into simian virus 40-transformed simian cells (COS-7), the transcript of the normal catalase/alpha-globin chimeric gene was spliced correctly, as revealed by Northern blotting and RNase mapping techniques. In contrast, the splicing of the mutant chimeric pre-mRNA occurred between the 5' donor site of the preceding intron and the 3' acceptor site of the intron containing the substitution, thereby skipping one entire exon sequence. Thus, the G to A transition at the fifth position of intron 4 of the catalase gene indeed severely limits the correct splicing of the RNA product. The same splice site mutation was found in the genomic DNA of another acatalasemic individual from an unrelated family. We suggest that this base substitution is the causal mutation of these cases of Japanese-type acatalasemia.

Acatalasia↗

PubMed1998

A splicing mutation of the RHAG gene associated with the Rhnull phenotype.

Rhnull is a syndrome serologically characterized by the deficiency of all Rh antigens on human red blood cells. Rhnull is divided into two types: regulator and amorph. Recently, Cherif-Zahar et al. proposed that the RHAG gene encoding the Rh50 glycoprotein is a candidate for inducing regulator type Rhnull. We investigated both the RH and RHAG genes in an Rhnull individual. The reticulocytes from the propositus had RHD, RHcE, and RHCe transcripts without any mutation. However, the sequence analysis of RHAG cDNA showed a deletion of 122 bp from nucleotide 946 to 1067. This deletion was revealed to be due to a homozygous splicing mutation, which is a single base substitution at the consensus sequence of the splicing acceptor site (AG-->AT). The mutation appeared to break the 'GT-AG' splicing rule and to cause 122 bp exon skipping accompanied by a frameshift. This study confirms that the RHAG gene is the most likely candidate for the 'regulator' gene of Rhnull cases.

Adolescent↗

PubMed2005

[Effect of a novel splicing mutation (IVS2-2A-->C) of SEDL gene on RNA processing].

X-linked spondyloepiphyseal dysplasia tarda (SEDL) is a rare osteochondrodysplasia caused by the mutation of SEDL gene, which mainly involves vertebral bodies and hips. To explore the effect of the novel splicing mutation (IVS2 -2A-->C) of SEDL gene on mRNA processing in a large Chinese family with X-linked spondyloepiphyseal dysplasia tarda and to elucidate the molecular base of SEDL, total RNA was isolated from EDTA blood samples of patients and controls. RT-PCR was performed on total RNA. cDNA was analyzed by bi-directionally direct sequencing of PCR products and Polyacrylamide gel electrophoresis (PAGE). Sequencing analysis revealed that there were two kinds of cDNA in patients. One is that exon 2 directly spliced exon 4, that is, exon 3 absence from the mature mRNA; and the other is that exon 1 directly spliced exon 4, meaning both exon 2 and 3 being spliced out completely. Meanwhile one kind of cDNA that exon 1 directly spliced exon 3 was found in normal controls. By PAGE, RT-PCR amplified products, 679bp and 537bp, were detected in normal controls, while 567bp and 425bp fragments were found in affected individuals. Our data show that the mutation of the splice-acceptor site in intron 2 causes exon 3 entirely exclusion from the mature RNA transcripts in affected individuals. As the translation start site of the SEDL gene locates on exon 3, the splicing defect causes affected individuals failure to produce sedlin, which elucidates the causative role of SEDL gene in the pathogenesis of SEDL. The absence of exon 2 indicates that there is alternative splicing in SEDL gene. The alternative splicing was also found in normal controls, which demonstrated that the alternative splicing might not be related to the phenotype of SEDL. Because the alternative splicing of exon 2 occurred in the 5'UTR, it is not clear whether it affects the gene expression.

Base Sequence↗

PubMed2003

Genomic definition of a pure intronic dystrophin deletion responsible for an XLDC splicing mutation: in vitro mimicking and antisense modulation of the splicing abnormality.

We characterised a dystrophin gene rearrangement in a previously described family with X-linked dilated cardiomyopathy and we demonstrated that it represents an 11 kb deletion occurring within intron 11. This unique deletion joined two physiologically distant intronic regions and brought adjacent two cryptic splice sites, generating a 159 bp sequence recognised as a novel alternative exon and spliced into the dystrophin transcript. Comparative analysis of the intronic region involved in the breakpoint revealed the presence of a LINE1 element (L1P_MA2), containing a 5' unconventional region (L1M1_5). This region provides the 5' cryptic splice site utilised by the novel exon, includes part of the region spliced into the dystrophin transcript and contains two short GA rich regions compatible with splicing motifs. We performed an in vitro splicing assay by using a minigene containing the patient minimal genomic rearrangement and we reproduced the inclusion of the novel alternative exon seen in the patient tissues. Antisense splicing modulation targeting the 3' cryptic splice site succeeded in restoring the canonical splicing. This represents a novel intronic mutational mechanism affecting the dystrophin gene and generating a splicing pathology. The definition of this mechanism might open perspectives in unravelling splicing regulatory motifs and their involvement in human genetic diseases.

Alternative Splicing↗

PubMed1999

Splicing mutations of 54-bp exons in the COL11A1 gene cause Marshall syndrome, but other mutations cause overlapping Marshall/Stickler phenotypes.

Stickler and Marshall syndromes are dominantly inherited chondrodysplasias characterized by midfacial hypoplasia, high myopia, and sensorineural-hearing deficit. Since the characteristics of these syndromes overlap, it has been argued whether they are distinct entities or different manifestations of a single syndrome. Several mutations causing Stickler syndrome have been found in the COL2A1 gene, and one mutation causing Stickler syndrome and one causing Marshall syndrome have been detected in the COL11A1 gene. We characterize here the genomic structure of the COL11A1 gene. Screening of patients with Stickler, Stickler-like, or Marshall syndrome pointed to 23 novel mutations. Genotypic-phenotypic comparison revealed an association between the Marshall syndrome phenotype and splicing mutations of 54-bp exons in the C-terminal region of the COL11A1 gene. Null-allele mutations in the COL2A1 gene led to a typical phenotype of Stickler syndrome. Some patients, however, presented with phenotypes of both Marshall and Stickler syndromes.

Abnormalities, Multiple↗

PubMed2006

New splicing mutation of MEN1 gene affecting the translocation of menin to the nucleus.

UNLABELLED: Multiple endocrine neoplasia type 1 (MEN1) is a syndrome inherited in an autosomal dominant trait caused by the inactivation of the tumor suppressor gene MEN1. OBJECTIVE: To communicate a family with a new heterozygous germ line mutation in the intronic region of MEN1 gene and to study its influence in the menin expression. PATIENTS AND METHODS: We studied 5 members of a family with symptomatic hyperparathyroidism (HPT). One of them had also a neuroendocrine pancreatic tumor, and 2 had non-functional multinodular cortical adrenal hyperplasia compatible with the diagnosis of MEN1. After the mutation was identified, HSP92II restriction enzyme was used to determine both zygosity and segregation of the mutation. RT-PCR from leukocyte's isolated mRNA and western blot from pancreatic tumor tissue were performed. In vitro studies were done in Chinese hamster ovary (CHO) cells transfected with reporter minigenes carrying the coding regions spanning exon (EX)-intron 9 and EX10 with the mutant and the wild type sequences. RESULTS: We identified a heterozygous G-to-T substitution in the intron-EX junction (IVS9-1 G>T) of MEN1 gene in the index case and the family members. The mRNA from patient's leukocytes was larger (934 bp) in comparison to the normal transcript (717 bp). Immunoblot analysis demonstrated that wild type (67 kDa) and two additional mutant proteins (approximately 55 and approximately 90 kDa) were expressed in the pancreatic tissue. The in vitro study showed a 45% nuclear localization of the mutated menin signal and a 95% in the wild type protein. CONCLUSIONS: We identified a new intronic heterozygous germ line mutation (IVS9-1G>T) of MEN1 gene in a family affected by MEN1 syndrome. This mutation alters the splice acceptor site of intron 9 that promotes an incorrect splicing, generating aberrant proteins without the nuclear localization signals necessary for the normal menin translocation to the nucleus.

Adult↗

PubMedDate not supplied

Clinical and mutational characterization of three patients with multiple sulfatase deficiency: report of a new splicing mutation.

Multiple sulfatase deficiency (MSD) is a rare autosomal recessive lysosomal storage disease characterized by impaired activity of all known sulfatases. The gene SUMF1, recently identified, encodes the enzyme responsible for post-translational modification of a cysteine residue, which is essential for the activity of sulfatases. Fewer than 30 MSD patients have been reported to date and 23 different mutations in the SUMF1 gene have been identified. Here, we present the characterization of the mutant alleles of two Spanish and one Argentinean MSD patients. While the two Spanish patients were homozygous for the previously described mutations, c.463T>C (p.S155P) and c.1033C>T (p.R345C), the Argentinean patient was homozygous for the new mutation IVS7+5 G>T. A minigene approach was used to analyze the effect of the splice site mutation identified, due to the lack of sample from the patient. This experiment showed that this change altered the normal splicing of the RNA, which strongly suggests that this is the molecular cause of the disease in this patient.

Base Sequence↗

PubMed1988

Splicing mutation in human hereditary analbuminemia.

We have identified a structural defect in the serum albumin gene in human analbuminemia. Sequence determination of 1.1 kilobases (kb) of the 5' regulatory region and of 6 kb across exonic regions revealed a single AG-to-GG mutation within the 3' splice site of intron 6 in the defective gene of an analbuminemic individual. In an in vitro assay on the RNA transcript this mutation causes a defect in splicing of the intron 6 sequence and in subsequent ligation of the exon 6-exon 7 sequences. Using polymerase-amplified genomic DNA and allele-specific oligodeoxynucleotide probes, we have also shown that the sequence of this intron 6/exon 7 splice junction is normal in a different, unrelated analbuminemic individual. Analbuminemia in humans is therefore the result of one of multiple defects in our genome.

Base Sequence↗