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At least 145 records · Page 8Linked to original sources
PubMed1990

Aberrant splicing of a naturally occurring alcohol dehydrogenase null activity allele in Drosophila melanogaster.

The DNA sequence of a naturally occurring alcohol dehydrogenase null activity allele, AdhnAC14, has eight extra nucleotides (in two groups of four) in the second intron, commencing six bases 3' from the 5' splice site. A stop codon was also found in exon 2. S1 nuclease protection experiments have shown that the insertions in intron 2 disrupt the correct splicing of intron 2. The null allele produces a transcript approximately 100 bases longer than the normal mature adult transcript, and the amount of the null allele transcript is only about 10% of the normal level.

Alcohol Dehydrogenase↗
PubMed2005

Type I Glanzmann thrombasthenia caused by an apparently silent beta3 mutation that results in aberrant splicing and reduced beta3 mRNA.

We report a novel genetic defect in a patient with type I Glanzmann thrombasthenia. Flow cytometry analysis revealed undetectable levels of platelet glycoproteins alphaIIb and beta3, although residual amounts of both proteins were detectable in immunoblotting analysis. Sequence analysis of reversely transcribed platelet beta3 mRNA showed a 100-base pair deletion in the 3'-boundary of exon 11, that results in a frame shift and appearance of a premature STOP codon. Analysis of the corresponding genomic DNA fragment revealed the presence of a homozygous C1815T transition in exon 11. The mutation does not change the amino acid residue but it creates an ectopic consensus splice donor site that is used preferentially, causing splicing out of part of exon 11. The parents of the proband, heterozygous for this mutation, were asymptomatic and had reduced platelet content of alphaIIbbeta3. PCR-based relative quantification of beta3 mRNA failed to detect the mutant transcript in the parents and showed a marked reduction in the patient. The results suggest that the thrombasthenic phenotype is, mainly, the result of the reduced availability of beta3-mRNA, most probably due to activation of the nonsense-mediated mRNA decay mechanism. They also show the convenience of analyzing both genomic DNA and mRNA, in order to ascertain the functional consequences of single nucleotide substitutions.

Alleles↗
PubMed1991

Maple syrup urine disease caused by a partial deletion in the inner E2 core domain of the branched chain alpha-keto acid dehydrogenase complex due to aberrant splicing. A single base deletion at a 5'-splice donor site of an intron of the E2 gene disrupts the consensus sequence in this region.

We have studied the molecular bases of maple syrup urine disease by analyzing the activity, subunit structure, mRNA sequence, and the genome of the affected enzyme. The branched chain alpha-keto acid dehydrogenase (BCKDH) activity in the patient was 4.2-4.5% of the control level. Immunoblot analysis revealed that the E2 subunit of BCKDH (Mr 52,000) was absent and another protein band with an Mr of 49,000 was present. We amplified the cDNA of the E2 subunit obtained from the patient's cell using the polymerase chain reaction method, then sequenced the amplified cDNA, in which a 78-bp deletion was identified. The consanguineous parents and a sister had two species of mRNA; the one corresponding to the normal E2 subunit and the other with a 78-bp deletion, whereas findings in a brother were normal. The molecular size of the translation products as deduced from the abnormal mRNA sequence was compatible with an abnormal protein band (Mr 49,000) detected in the patient's cells by immunoblot analysis. Analysis of genomic DNA of BCKDH-E2 subunit revealed that the 78-bp deletion in the mRNA was caused by an exon skipping due to a single base deletion in the 5'-splice donor site. As a result of the mutation, part of the inner E2 core domain was omitted. The specified region of the inner E2 core domain was highly homologous to the region of the E2 subunit of pyruvate dehydrogenase and alpha-ketoglutarate dehydrogenase. These observations imply the biological importance of the region in the inner E2 core domain of BCKDH to maintain normal function of the activity.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1993

Carbamyl phosphate synthetase I deficiency. One base substitution in an exon of the CPS I gene causes a 9-basepair deletion due to aberrant splicing.

Carbamyl phosphate synthetase I (CPS I; EC6,3,4,16) is an autosomal recessive disorder characterized by hyperammonemia. We studied the molecular bases of CPS I deficiency in a newborn Japanese girl with consanguineous parents. Northern and Western blots revealed a marked decrease in CPS I mRNA and enzyme protein but with a size similar to that of the control, respectively. Sequencing of the patient's cDNA revealed a nine-nucleotide deletion at position 832-840. Sequencing analysis of the genomic DNA revealed a G to C transversion at position 840, the last nucleotide of an exon in the splice donor site. This substitution altered the consensus sequence of the splice donor site and the newly cryptical donor site in the exon caused the 9-bp in-frame deletion. This report seems to be the first complete definition of CPS I deficiency, at the molecular level.

Amino Acid Sequence↗
PubMed2004

A Novel IVS2-1G>A mutation causes aberrant splicing of the HRPT2 gene in a family with hyperparathyroidism-jaw tumor syndrome.

HRPT2, the gene associated with hyperparathyroidism-jaw tumor (HPT-JT) syndrome, was previously mapped to 1q24-q32. It was recently cloned, and several germline mutations were found to predispose to HPT-JT syndrome. We sequenced the complete HRPT2 coding sequence and splice-junctional regions in a Korean family with HPT-JT syndrome and identified a novel germline mutation, IVS2-1G>A in intron 2, that caused the autosomal dominant trait of HPT-JT syndrome in this family. RT-PCR and sequencing of the transcripts revealed that this splicing mutation generated alternative splicing errors leading to the formation of two different transcripts, one with exon 3 deleted, the other lacking the first 23 bp of exon 3 due to the use of an internal splice acceptor in exon 3. Translation of both transcripts results in premature termination. In addition, we detected two novel somatic mutations of HRPT2 in malignant parathyroid tumors from the affected individuals. One, 85delG, causes premature termination; the other, an 18 bp in-frame deletion of 13_30delCTTAGCGTCCTGCGACAG, suggests that this region may be important in the development of the parathyroid carcinomas in HPT-JT syndrome. These findings provide further evidence that mutation of HRPT2 is associated with the formation of parathyroid tumors in HPT-JT syndrome.

Adult↗
PubMed1999

Aberrant splicing in the PKD2 gene as a cause of polycystic kidney disease.

It is estimated that approximately 15% of families with autosomal dominant polycystic kidney disease (ADPKD) have mutations in PKD2. Identification of these mutations is central to identifying functionally important regions of gene and to understanding the mechanisms underlying the pathogenesis of the disorder. The current study describes mutations in six type 2 ADPKD families. Two single base substitution mutations discovered in the ORF in exon 14 constitute the most COOH-terminal pathogenic variants described to date. One of these mutations is a nonsense change and the other encodes an apparent missense variant. Reverse transcription-PCR from patient lymphoblast RNA showed that, in addition, both mutations resulted in out-of-frame splice variants by activating cryptic splice sites via different mechanisms. The apparent missense variant produced such a strong splicing signal that the processed transcript from the mutant chromosome did not contain any of the normally spliced, missense product. A third mutation, a nonconservative missense change effecting a negatively charged residue in the third transmembrane span, is likely pathogenic and defines a highly conserved residue consistent with a potential channel subunit function for polycystin-2. The remaining three mutations included two frame shifts resulting from deletion of one or two bases in exons 6 and 10, respectively, and a nonsense mutation due to a single base substitution in exon 4. The study also defined a novel intragenic polymorphism in exon 1 that will be useful in analyzing "second hits" in PKD2. Finally, the study demonstrates that there are reduced levels of normal polycystin-2 protein in lymphoblast lines from PKD2-affected individuals and that truncated mutant polycystin-2 cannot be detected in patient lymphoblasts, suggesting that the latter may be unstable in at least some tissues. The mutations described will serve as critical reagents for future functional studies in PKD2.

Humans↗
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↗
PubMed1995

An aberrant splicing using a 3' cryptic splice site within the CH1 exon induces truncated mu-chain production.

AT8--1-12--5-1, an Abelson virus-transformed immature B-cell line, produced truncated mu-chains. Sequencing analysis of the mu-expressed allele revealed that the variable region was an out-of-frame VH7183-DSP2-JH3 complex. Two cDNA clones (5-1 cDNA1 and 5-1 cDNA2) derived from the transcripts of the mu-expressed allele were cloned and sequenced. Sequencing analysis of 5-1 cDNA1 revealed that the VH7183-DSP2-JH3 sequence jointed to the CH1 exon at 136 bp, 3' from the 5' end of the CH1 exon, resulting in the change of the reading frame from out-of-frame to in-frame. On the other hand, sequencing analysis of 5-1 cDNA2, which appeared to have derived from intron-containing premature mRNA, revealed that the J-C intron sequence joined to the CH1 exon at 110 bp 3' from the 5' end of the CH1 exon, indicating the deletion of 109 bp including the 3' splice site of the CH1 exon. These results demonstrate that the deletion of the authentic 3' splice site of the CH1 exon induced activation of the cryptic splice site within the CH1 exon. This was followed by splicing of the variable region to the CH1 exon at the cryptic splice site at 136 bp 3' from the 5' end of the CH1 exon, resulting in the change of the reading frame from out-of-frame to in-frame, followed by the truncated mu-chain production.

Alleles↗
PubMed1993

Frequent activation of the lck gene by promoter insertion and aberrant splicing in murine leukemia virus-induced rat lymphomas.

We have analysed DNA and RNA from 36 T-cell lymphomas induced in Fischer rats by Moloney murine leukemia virus for alterations affecting the structure or expression of the lck gene. At least five primary tumors (14%) have a proviral insertion upstream of lck. In at least four of the tumors, proviral insertion increases lck mRNA levels an average of eight-fold. Overexpression of lck results from transcription initiating in the viral promoter and extending into lck sequences. Three different structures of hybrid transcript were detected. In all three, the hybrid RNAs are spliced to a normal lck splice acceptor in the first exon of lck, resulting in removal of three out of frame ATG codons which would be expected to increase the translation efficiency of the hybrid message. In one tumor, the viral splice donor is used, in one tumor, proviral insertion generates a splice donor sequence one base pair downstream of the long terminal repeat boundary, and in two tumors, a cryptic splice donor in the upstream lck sequences is used. The significance of these unusual splicing patterns and of the higher frequency of proviral insertions adjacent to lck in rats relative to mice is discussed.

Animals↗
PubMed1998

High level accumulation of an aberrantly spliced human DHFR RNA species.

Cells transduced with either of two human DHFR minigenes express an RNA product which is considerably shorter than what would be predicted from the size of an unspliced transcript expressed from its DNA template. RNA blotting analysis has shown that this short transcript accumulated to exceedingly high levels which were comparable to the levels reached by the highly abundant endogenous actin mRNA, or MoMLV RNA expressed in chronically infected cells. RNA blotting, RNase mapping, primer extension, RT-PCR, and sequencing have shown that this highly expressed transcript, termed TBN, is a spliced RNA product which utilizes cryptic splice signals present in the normally spliced DHFR mRNA. Subcloning experiments have demonstrated that all the information required for the generation and high level accumulation of the TBN transcripts is encoded in the 1.6 kb DHFR DNA minigene. TBN transcripts were generated with comparable efficiency from DNA templates containing either the human ADA or the early SV40 promoters. Since neither the ADA nor the SV40 promoter are considered to be particularly "strong" promoters, this observation argues that initiation of transcription is not the rate limiting step in determining the amount of the TBN transcripts which accumulate in the cell. Insertion of a foreign sequence into the DHFR DNA minigene led to the expression and high level accumulation of a chimeric transcript, suggesting that the unusual properties of this expression system may be used for high level expression of foreign sequences. These observations offer new insights into the mechanisms which control the accumulation of translatable mRNA in the cell, and have potentially important implications for experiments involving optimization of gene expression for gene therapy applications.

3T3 Cells↗
PubMed2005

Functional significance of a deep intronic mutation in the ATM gene and evidence for an alternative exon 28a.

Screening for ATM mutations is usually performed using genomic DNA as a template for PCR amplification across exonic regions, with the consequence that deep intronic sequences are not analyzed. Here we report a novel pseudoexon-retaining deep intronic mutation (IVS28-159A>G; g.75117A>G based on GenBank U82828.1) in a patient with ataxia-telangiectasia (A-T), as well as the identification of a previously unrecognized alternative exon in the ATM gene (exon 28a) expressed in lymphoblastoid cell lines (LCL) derived from normal individuals. cDNA analysis using the A-T patient's LCL showed the retention of two aberrant intronic segments of 112 and 190 nt between exons 28 and 29. Minigenes were constructed to determine the functional significance of two genomic changes in the region of aberrant splicing: IVS28-193C>T (g.75083C>T) and IVS28-159A>G, revealing that: 1) the first is a polymorphism; 2) IVS28-159A>G weakens the 5' splice site of the alternative exon 28a and activates a cryptic 5' splice site (ss) 83 nt downstream; and 3) wild-type constructs also retain a 29-nt segment (exon 28a) as part of both the 112- and 190-nt segments. Maximum entropy estimates of ss strengths corroborate the cDNA and minigene findings. Such mutations may prove relevant in planning therapy that targets specific splicing aberrations.

Alternative Splicing↗
PubMed2003

Protocol for the sequence analysis of ryanodine receptor subtype 1 gene transcripts from human leukocytes.

BACKGROUND: The search for novel mutations in the ryanodine receptor subtype 1 (RYR1) gene causing malignant hyperthermia and central core disease is hampered by the fact that the gene contains 106 exons. Searching for novel mutations in complementary DNA (cDNA) requires an invasive muscle biopsy. Accordingly, an alternate source of RYR1 cDNA was sought for sequence analysis. METHODS: Leukocytes were isolated from human blood and used for extraction of RNA and reverse transcription of messenger RNA into cDNA. A detailed protocol was developed in which overlapping fragments of RYR1 cDNA were amplified by polymerase chain reaction in a series of steps and used for double-strand sequencing. RESULTS: The sequences of full-length leukocyte RYR1 cDNA obtained from four human blood samples were shown to be identical to the sequence of a human muscle RYR1 cDNA. The incidence of aberrant splicing was more pronounced in the blood-derived cDNAs, but this could be minimized by adequate sample preparation. Protocols to sequence alternatively spliced products were also developed. Several silent nucleotide polymorphisms were detected, and minor revisions were made to the RYR1 sequence. CONCLUSIONS: Because there are no differences in RYR1 transcript structure between muscle and leukocytes, aside from those that may be ascribed to RNA splicing aberrations during processing, leukocytes seem to be an adequate substitute tissue for screening the RYR1 gene for previously undiscovered mutations in families with malignant hyperthermia or central core disease.

Base Sequence↗
PubMed1996

Aberrant RNA splicing of the protein C and protein S genes in healthy individuals.

RNA-based studies are an important tool for the identification and functional characterization of mutations underlying inherited disease. These studies could in principle be compromised by 'aberrant splicing' (the generation of alternatively spliced transcripts lacking any obvious function) during normal expression of the genes under investigation. Using a highly sensitive RT-PCR assay, we show here that aberrant splicing is a frequent occurrence during expression of the protein C (PROC) and protein S (PROS) genes. Aberrantly spliced transcripts were present in different cell types including liver, the main expressing tissue for both protein C and protein S. In an attempt to compare individual mRNA splicing patterns, PROC and PROS RNA from easily accessible cells of different healthy control individuals was studied. However, variation between different RT-PCR assays from the same individual precluded both the relative quantitation of the aberrant transcripts and the analysis of interindividual differences. Our findings are consistent with the notion that a low level of aberrantly spliced transcripts are routinely generated during PROC and PROS gene expression. The possibility that these transcripts may complicate the RT-PCR analysis of pathological transcripts must be taken into account when RNA-based strategies of disease analysis are considered.

Electrophoresis, Agar Gel↗
PubMed2005

Single-nucleotide polymorphisms in NAGNAG acceptors are highly predictive for variations of alternative splicing.

Aberrant or modified splicing patterns of genes are causative for many human diseases. Therefore, the identification of genetic variations that cause changes in the splicing pattern of a gene is important. Elsewhere, we described the widespread occurrence of alternative splicing at NAGNAG acceptors. Here, we report a genomewide screen for single-nucleotide polymorphisms (SNPs) that affect such tandem acceptors. From 121 SNPs identified, we extracted 64 SNPs that most likely affect alternative NAGNAG splicing. We demonstrate that the NAGNAG motif is necessary and sufficient for this type of alternative splicing. The evolutionarily young NAGNAG alleles, as determined by the comparison with the chimpanzee genome, exhibit the same biases toward intron phase 1 and single-amino acid insertion/deletions that were already observed for all human NAGNAG acceptors. Since 28% of the NAGNAG SNPs occur in known disease genes, they represent preferable candidates for a more-detailed functional analysis, especially since the splice relevance for some of the coding SNPs is overlooked. Against the background of a general lack of methods for identifying splice-relevant SNPs, the presented approach is highly effective in the prediction of polymorphisms that are causal for variations in alternative splicing.

Alleles↗
PubMed1997

Aberrant mRNA splicing causes sorbitol dehydrogenase deficiency in C57BL/LiA mice.

Sorbitol dehydrogenase (Sord) catalyzes the interconversion of sorbitol and fructose and is functionally important both in the metabolism of dietary sorbitol and as a source of fructose in semen. Together with aldose reductase, Sord forms the polyol pathway, which plays an important role in the etiology of diabetic complications. The Sord-deficient mouse (C57BL/ LiA) is very useful in animal model studies of the involvement of the polyol pathway in both diabetic and congenital cataracts. To understand more about this strain, we characterized the molecular basis underlying this Sord deficiency and found that this was due to a point mutation in the exon 8/intron 8 junction. Substitution of an A for G at the first position of the strictly conserved GT donor completely abolished normal splicing of exon 8. Aberrant splicing of this junction generates at least three types of transcripts: one lacking exon 8, another that has a truncated exon 8, and a third that contains intron sequences. We have devised two convenient PCR-based methods to identify this mutation in C57BL/LiA mice. These methods are useful in animal experiments that involve cross-breeding with these mice because they allow early determination of genotype without the need to sacrifice the animals for enzyme assay.

Alleles↗
PubMed2004

5'CpG island hypermethylation and aberrant transcript splicing both contribute to the inactivation of the FHIT gene in resected non-small cell lung cancer.

The relative contribution of promoter hypermethylation and aberrant splicing to the inactivation of the fragile histidine triad (FHIT) gene is unclear. Using genetic and epigenetic analyses, the current investigation examines the loss of protein and mRNA expression, and 5'CpG hypermethylation and allelic imbalance of the FHIT gene in a series of 129 non-small cell lung cancer (NSCLC) samples, in parallel with clinicopathological analyses. We found that 50% of NSCLC patients had aberrant protein expression, which was more frequent in squamous cell carcinomas (SQ) (69%) than in adenocarcinomas (AD) (28%) (P < 0.0001). 5'CpG hypermethylation of FHIT was identified in 31% of patients. Abnormally-sized FHIT transcripts were also observed in 24% of patients and were attributed to various exonic deletions, mainly in the region of exons 4-8. Allelic imbalance of the FHIT locus and its correlation with the status of Fhit expression, 5'CpG hypermethylation, and aberrant splicing, indicated that biallelic inactivation of Fhit expression could be induced by 5'CpG hypermethylation of one allele and alternative splicing in the other allele. Moreover, an 83% concordance in the methylation status of FHIT was demonstrated between 12 samples of bronchial precancerous lesions taken before surgery and their matched resected tumours. Our data suggest that FHIT 5'CpG hypermethylation and splicing alterations are both predominant mechanisms involved in the aberrant expression of the FHIT gene, and that FHIT 5'CpG methylation may be potentially used as a supplemental detection marker for NSCLC.

Acid Anhydride Hydrolases↗