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At least 523 records · Page 29Linked to original sources
PubMed1993

A splice junction mutation causes deletion of a 72-base exon from the mRNA for lysosomal acid lipase in a patient with cholesteryl ester storage disease.

The genetic defect leading to cholesteryl ester storage disease (CESD) has been determined in a 12-yr-old patient. Lysosomal acid lipase (LAL) activity in cultured skin fibroblasts was reduced to approximately 9% of control fibroblasts. Plasma cholesterol (255 mg/dl) and LDL-cholesterol (215 mg/dl) were elevated whereas HDL-cholesterol was reduced (19 mg/dl). Triglycerides were moderately elevated (141 mg/dl). There were no clinical abnormalities with the exception of hepatosplenomegaly. Both parents have reduced LAL activity in white blood cells. PCR analysis of the LAL mRNA from the propositus revealed a single slightly smaller mRNA species in skin fibroblasts as well as in leukocytes. The mother of the patient and his older brother had two mRNA species: one of normal size and one of the same size as the propositus. The father has a LAL mRNA of normal size only. Sequence analysis of a PCR-amplified cDNA fragment showed a 72-bp in-frame deletion resulting in the loss of the codons for amino acids 254-277. Analysis of genomic DNA revealed that the 72 bp represent an exon, indicating that the deletion in the mRNA is caused by defective splicing. Sequence analysis of the patient's genomic DNA revealed a G-->A substitution in the last nucleotide of the 72-bp exon in one of his alleles. The mutant allele was shown to cosegregate with the truncated mRNA in the pedigree, providing further evidence that the G-->A substitution causes aberrant splicing and exon skipping. No normal-sized mRNA is detectable in the propositus even though he is not homozygous for the splice site mutation. This can be only accounted for by assuming that he is a compound heterozygote with a null allele inherited from his father. In summary, the data presented provide evidence that deletion of the codons for amino acids 254-277 in the LAL mRNA in combination with a null allele cause the clinical expression of CESD in our patient.

Adolescent↗
PubMed1996

The mechanism of protein splicing and its modulation by mutation.

Protein splicing results in the expression of two mature proteins from a single gene. After synthesis of a precursor protein, an internal segment (the intein) is excised and the external domains are joined together. A self-catalyzed mechanism for this cleavage-ligation reaction is presented, based on mutagenesis data and analysis of splicing intermediates. Mutations were used to block various steps in the protein splicing pathway, allowing each isolated step to be studied independently. A linear ester intermediate was identified and functional roles for the four conserved splice junction residues were determined. Understanding the mechanism of protein splicing provides a basis for protein engineering studies. For example, inteins can be constructed which fail to splice, but instead cleave the peptide bond at a chosen splice junction.

Bacterial Proteins↗
PubMed1996

Germinal HPRT splice donor site mutation results in multiple RNA splicing products in T-lymphocyte cultures.

We have used peripheral blood T-lymphocyte cultures to analyze the hprt mutation in two Lesch-Nyhan syndrome males who are cousins and to confirm the carrier status of female members of the family. Both cDNA and genomic DNA sequencing studies show that this patient carries a hitherto undescribed single base deletion in the exon 5 donor splice site sequence (I5: +1, delta G, base number 31635). The largest cDNA product contained all nine hprt exons plus an insertion of 66 bases of intron 5, consistent with the use of a cryptic splice site in intron 5 (aag67/gtaagc). This splicing error would result in a chain terminating codon immediately after exon 5 (I5:2-4, taa) and predicts a polypeptide of 133 amino acids. This loss of the normal splice donor site also results in multiple hprt mRNA species, combining the use of the cryptic splice site in intron 5 and splicing errors involving exons 2-6. In addition to defining a new Lesch-Nyhan mutation (hprtHenryville), these results provide insight into aberrant splicing of hprt mRNA in T-lymphocytes.

Base Sequence↗
PubMed1993

COL4A5 splice site mutation and alpha 5(IV) collagen mRNA in Alport syndrome.

Mutations affecting the COL4A5 gene encoding the alpha 5 chain of type IV collagen, are involved in the pathogenesis of X-linked Alport syndrome. We used denaturing gradient gel electrophoresis (DGGE) to screen PCR amplified exons of COL4A5 for point mutations in a set of 18 Alport patients previously characterized by Southern blotting. One sequence variant was identified in the exon 38 region of a male Alport patient. Sequence analysis revealed a G to C transversion in the 5' intron splice donor site downstream from exon 38 (GT to CT). To determine the effect of the mutation on mRNA splicing, alpha 5(IV) cDNA was generated from total RNA of peripheral blood lymphocytes. Subsequent cDNA PCR yielded a product 81 base pairs shorter in the affected Alport patient, compared to normal controls. The absence of exon 38 from the alpha 5(IV) cDNA was confirmed by sequence analysis. The results demonstrated that the mutation leads to skipping of exon 38 in the processing of alpha 5(IV) pre-mRNA. The shortened transcript lacked 27 codons encoding a Gly-X-Y-repeat sequence with a preserved reading frame, enabling the translation of codons further downstream. Clinically, the patient presented with juvenile onset Alport syndrome, end-stage renal failure, and deafness. He had no ocular lesions. Typical ultrastructural changes of the glomerular basement membrane (GBM) were shown on electron microscopy. The patient developed anti-GBM antibodies after renal transplantation, however, renal function deteriorated only moderately.

Adult↗
PubMed1996

Gene defects in congenital factor XIII deficiency.

The knowledge of the molecular basis of factor XIII deficiency has improved significantly in recent years. Almost 20 different mutations have been described in the gene coding for the factor A-subunit and 3 mutations in the gene coding for the B-subunit. Half of the mutations in the factor XIIIa A-subunit gene are nonsense mutations that result in premature termination of translation. Three of them are frameshift mutations that are caused by minor deletions. Two of them are splicing mutations and 3 are stop mutations that are caused by single nucleotide substitutions. Ten of the mutations are missense mutations caused by nucleotide transitions leading to amino acid substitutions. In the factor XIII B-subunit gene, the 3 mutations are an amino acid substitution, a splicing mutation, and a trinucleotide insertion. These mutations explain the disease in the two families reported to have XIII B-subunit deficiency. In factor XIII A-subunit deficiency, the genetic defects have been characterized so far only in a minority of cases. In most of the reports of factor XIII A-subunit mutations, each family carries its own mutation/mutations. However, in some populations such as Finns and Arabs some enrichment of specific mutations has occurred. Some international migration of a few mutations has also been noted. The structural and functional effects of the mutations have been analyzed by studying the expression of the factor XIII subunits on mRNA and protein levels in vivo or in vitro, and by utilizing the three-dimensional model of crystallized factor XIII A-subunit in modeling of the missense mutations.

Arabs↗
PubMed1999

Hepatosplenomegalic lipidosis: what unless Gaucher? Adult cholesteryl ester storage disease (CESD) with anemia, mesenteric lipodystrophy, increased plasma chitotriosidase activity and a homozygous lysosomal acid lipase -1 exon 8 splice junction mutation.

A 36-year-old woman was admitted for hepatosplenomegaly and anemia. Bone marrow cytology showed "sea-blue histiocytes", vacuolated macrophages and plasma cells. As primary liver disease, malignancy or hematologic disorders were excluded, and plasma chitotriosidase activity was increased 27-fold over control, the presence of a lysosomal storage disease was suspected. Biochemical analysis of skin fibroblasts revealed normal glucocerebrosidase and sphingomyelinase activity, but lipid analysis showed a more than 15-fold accumulation of cholesterol esters within the cells. The activity of lysosomal acid lipase (LAL) in fibroblast homogenates was decreased to 12% of control subjects. Mutational analysis of the patient's blood showed the homozygous G-->A mutation at position -1 of the exon 8 splice donor site (E8SJM-allele) known for adult cholesteryl ester storage disease (CESD); the polymorphic background was that of the complex haplotype -6Thr, 2Gly, 894 G-->A. Based on clinical, laboratory, cytological and and biochemical findings, CESD can clearly be separated from other more frequent inherited lysosomal storage diseases, e.g. atypical forms of Gaucher disease.

Adult↗
PubMed1999

A heterozygous splice site mutation in COL6A1 leading to an in-frame deletion of the alpha1(VI) collagen chain in an italian family affected by bethlem myopathy.

Bethlem myopathy is a mild neuromuscular disorder with proximal muscular weakness and early flexion contractures. It is an autosomal dominant disease due to mutations in type VI collagen genes. We found a T-->C substitution at the +2 position of COL6A1 intron 14 in a family, leading to skipping of exon 14 and an in-frame deletion of 18 amino acids in the triple-helical domain of the alpha1(VI) collagen chain. The deletion included a cysteine residue believed to be involved in the assembly of type VI collagen dimers intracellularly, prior to the protein secretion. Analysis of the affected fibroblasts showed that the shortened alpha1(VI) collagen chains were synthesized but not secreted by the cells and that the amount of type VI collagen microfibrils deposited by the cells was reduced. The results suggest that the clinical phenotype is due to a reduction in the level of type VI collagen in the extracellular matrix.

Adult↗
PubMed1999

Donor splice site mutation in keratin 5 causes in-frame removal of 22 amino acids of H1 and 1A rod domains in Dowling-Meara epidermolysis bullosa simplex.

Epidermolysis bullosa simplex (EBS) arises from mutations within the keratin 5 and 14 (K5 and K14) genes which alter the integrity of basal keratinocytes cytoskeleton. The majority of these defects are missense mutations in the rod domain, whose locations influence the disease severity. We investigated a large family dominantly affected with the Dowling-Meara form of EBS (EBS-DM). Sequencing of amplified and cloned K5 cDNA from cultured keratinocytes revealed a 66 nucleotide deletion in one allele corresponding to the last 22 amino acid residues encoded by exon 1 (Val164 to Lys185). Sequencing of amplified genomic DNA spanning the mutant region revealed a heterozygous G-to-A transition at +1 position of the consensus GT donor splice site of intron 1 of K5. This mutation leads to the use of an exonic GT cryptic donor splice site, located 66 nucleotides upstream from the normal donor splice site of intron 1. The corresponding peptide deletion includes the last five amino acids of the H1 head domain and the first 17 amino acids of the conserved amino terminal end of the 1A rod domain, including the first two heptad repeats and the helix initiation peptide. The shortened polypeptide is expressed in cultured keratinocytes at levels which are comparable to the normal K5 protein. This is the first splice site mutation to be reported as a cause of EBS-DM. Owing to the functional importance of the removed region, our data strongly suggest that shortened keratin polypeptide can impair keratin filament assembly in a dominant manner and causes EBS-DM.

Alternative Splicing↗
PubMed1998

Exon 1 donor splice site mutations in the porphobilinogen deaminase gene in the non-erythroid variant form of acute intermittent porphyria.

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial defect of the heme biosynthesis enzyme, porphobilinogen deaminase (PBGD). PBGD is encoded by two distinct mRNA species expressed in a tissue-specific manner from a single gene. One transcript is expressed in erythroid tissues, while the housekeeping transcript is expressed in all tissues. In classical AIP (95% of cases) the housekeeping and the erythroid-specific enzymes both have half-normal activity in erythroid and non-erythroid tissues, whereas in the variant non-erythroid form of the disease the enzymatic defect is present only in non-erythroid cells. A large allelic heterogeneity of mutations (n>135) has been demonstrated in classical AIP, but to date only three different mutations have been characterized in the non-erythroid variant form of AIP. We describe the molecular abnormalities responsible for the non-erythroid variant form of AIP in two French and two German unrelated AIP patients with normal PBGD activity in the erythrocytes. Three different splicing defects located in the intron 1 donor splice site were identified: a 33+1 g-->a mutation, previously described in a Dutch family, was found in two patients; two novel mutations (33+2 t-->a, 33+5 c-->g) affected the two remaining patients. All the mutations resulted in the activation of a cryptic splice site 67 bp downstream in intron 1, leading to a frameshift and a premature stop codon in exon 4. Mutations in the exon 1 donor splice site are involved in eight of the nine non-erythroid variant AIP families reported in the literature. These data show that most mutations causing the non-erythroid variant AIP are exon 1 splice defects, in contrast with classical AIP, where missense mutations are chiefly involved. Moreover, the allelic heterogeneity of PBGD gene defects causing the non-erythroid variant AIP is demonstrated, with five different mutations identified. These mutations could be easily detected by a single denaturing gradient gel electrophoresis which also allows the presymptomatic detection of gene carriers in the affected families.

Adult↗
PubMed1993

A 5' splice site mutation in fucosidosis.

Fucosidosis is a rare, autosomal recessive, lysosomal storage disease, resulting from a deficiency of the enzyme alpha-fucosidase (EC 3.2.1.51). It is characterised clinically by progressive mental and motor deterioration, growth retardation, coarse facies, and often recurrent infections, but the course of the disease is variable. The gene encoding lysosomal alpha-fucosidase has been mapped to the short arm of chromosome 1 at position 1p34.1-36.1 and has been called FUCA1. Two mutations causing disease have been described previously, a C-->T change in exon 8 giving rise to a premature, in frame TAA stop codon, and a deletion of at least two exons from the 3' end of the gene. In this paper we present evidence that a homozygous G-->A transition in the first position of the 5' splice site of intron 5 of FUCA1 is the disease causing mutation in a 9 year old child of distantly related parents. A new banding pattern was detected in the patient by Southern blotting of genomic DNA using TaqI restriction and a cDNA FUCA1 probe. The patient was homozygous for this pattern. Three sibs with alpha-fucosidase activity below the normal reference range and both parents were heterozygous. This pattern was not detected in 26 other fucosidosis patients and has not been found in any controls. The mutation was localised by a combination of restriction mapping using different cDNA probes, single stranded conformational polymorphism analysis of exons and flanking regions amplified by the polymerase chain reaction, and by direct sequencing of the amplified sequence. A view of the nature of the mutation, its cosegregation with the disease mutation and its absence in controls, it is probable that the 5' splice site mutation causes fucosidosis in this child.

Base Sequence↗
PubMed1986

Adenovirus mutants with splice-enhancing mutations in the E3 complex transcription unit are also defective in E3A RNA 3'-end formation.

Region E3 of adenovirus encodes about 10 overlapping mRNAs with different spliced structures. The mRNAs are 5' coterminal and form two major 3'-coterminal families termed E3A and E3B. As a group, the mRNAs have two 5' splice sites and four or five 3' splice sites. We previously described a novel class of virus mutants with deletions that enhance distant upstream and downstream 5' and 3' splice sites in region E3 (S. L. Deutscher, B. M. Bhat, M. H. Pursley, C. Cladaras, and W. S. M. Wold, Nucleic Acids Res. 13:5771-5788, 1985). We now report that two of these mutants, dl710 and dl712, are defective in RNA 3'-end formation at the E3A site. This result was surprising because the deletions in dl710 and dl712 are upstream of the putative signal for E3A RNA 3'-end formation. The explanation that we favor for this result is that the enhanced splicing activity in these mutants results in the splicing out of the E3A 3'-end site from the RNA precursor before the E3A 3' ends have a chance to form.

Adenoviridae↗
PubMed2005

A unique exonic splice enhancer mutation in a family with X-linked mental retardation and epilepsy points to a novel role of the renin receptor.

The renin-angiotensin system (RAS) is essential for blood pressure control and water-electrolyte balance. Until the discovery of the renin receptor, renin was believed to be mainly a circulating enzyme with a unique function, the cleavage of angiotensinogen. We report a unique mutation in the renin receptor gene (ATP6AP2) present in patients with X-linked mental retardation and epilepsy (OMIM no. 300423), but absent in 1200 control X-chromosomes. A silent mutation (c.321C>T, p.D107D) residing in a putative exonic splicing enhancer site resulted in inefficient inclusion of exon 4 in 50% of renin receptor mRNA, as demonstrated by quantitative RT-PCR. Analysis of membrane associated-receptor molecular forms showed the presence of full-length and truncated proteins in the patient. Functional analysis demonstrated that the mutated receptor could bind renin and increase renin catalytic activity, similar to the wild-type receptor, but resulted in a modest and reproducible impairment of ERK1/2 activation. Thus, our findings confirm the importance of the RAS in cognitive processes and indicate a novel specific role for the renin receptor in cognitive functions and brain development.

Alternative Splicing↗
PubMed1999

Identification of a 5' splice site mutation in the RPGR gene in a family with X-linked retinitis pigmentosa (RP3).

We have identified a novel RPGR gene mutation in a large Dutch family with X-linked retinitis pigmentosa (RP3). In affected members, a G-->T transversion was found at position +1 of the 5' splice site of intron 5 of the RPGR (retinitis pigmentosa GTPase regulator) gene. Analysis of this mutation at the RNA level showed cryptic splicing upstream of the mutation in exon 5 leading to a frameshift and downstream termination codon. Identification of the causative mutation in this family has facilitated the detection of females at risk of having an affected son.

Carrier Proteins↗
PubMed2001

Niemann-Pick disease type C: spectrum of HE1 mutations and genotype/phenotype correlations in the NPC2 group.

In Niemann-Pick disease type C (NPC), a genetic heterogeneity with two complementation groups--NPC1, comprising > or =95% of the families, and NPC2--has been demonstrated. Mutations in the NPC1 gene have now been well characterized. HE1 was recently identified as the gene underlying the very rare NPC2. Here we report the first comprehensive study of eight unrelated families with NPC2, originating from France, Algeria, Italy, Germany, the Czech Republic, and Turkey. These cases represent essentially all patients with NPC2 who have been reported in the literature, as well as those known to us. All 16 mutant alleles were identified, but only five different mutations, all with a severe impact on the protein, were found; these five mutations were as follows: two nonsense mutations (E20X and E118X), a 1-bp deletion (27delG), a splice mutation (IVS2+5G-->A), and a missense mutation (S67P) resulting in reduced amounts of abnormal HE1 protein. E20X, with an overall allele frequency of 56%, was established as the common mutant allele. Prenatal diagnosis was achieved by mutation analysis of an uncultured chorionic-villus sample. All mutations except 27delG were observed in a homozygous state, allowing genotype/phenotype correlations. In seven families (with E20X, E118X, S67P, and E20X/27delG mutations), patients suffered a severe and rapid disease course, with age at death being 6 mo-4 years. A remarkable feature was the pronounced lung involvement, leading, in six patients, to early death caused by respiratory failure. Two patients also developed a severe neurological disease with onset during infancy. Conversely, the splice mutation corresponded to a very different clinical presentation, with juvenile onset of neurological symptoms and prolonged survival. This mutation generated multiple transcripts, including a minute proportion of normally spliced RNA, which may explain the milder phenotype.

Adult↗
PubMed1994

Metachromatic leukodystrophy in the Navajo Indian population: a splice site mutation in intron 4 of the arylsulfatase A gene.

Metachromatic leukodystrophy (MLD) is an autosomal recessive disorder of myelin metabolism, resulting from the inability to properly degrade 3-sulfogalactosylceramide (sulfatide). This metabolic block is often due to defective functioning of the lysosomal enzyme arylsulfatase A (ARSA). Unmetabolized sulfatide accumulates in the white matter of the CNS and in the peripheral nerves, leading to progressive demyelination and death. Late infantile, juvenile and adult clinical variants of MLD have been described. A Navajo Indian child was diagnosed with late infantile MLD (LIMLD), and his ARSA gene was amplified in three overlapping regions by the PCR and sequenced. A single mutation was found: a G-->A transition in the first nucleotide of intron 4 (IVS4nt1), which abolishes the 5' splice site consensus sequence. Negligible amounts of ARSA mRNA were observed in Northern blots. However, PCR amplification and sequencing of the ARSA cDNA showed that all of the mRNA species from the patient have exon 4 deleted. A new reading frame is thus established which results in a premature stop codon within exon 5. A minority of transcripts had additional splicing errors. Both parents carry this mutation, and the father also carries the pseudodeficiency (PD) allele. Three additional unrelated Navajo LIMLD patients were found to be homozygous for the same MLD-causing mutation by allele-specific oligonucleotide (ASO) hybridization. This method could be used for carrier and patient identification in this population.

Adult↗