Serum levels of Clara cell 10-kDa protein are decreased in patients with asthma

The human immunodeficiency virus type-1 regulatory protein Rev is absolutely required for the production of viral structural proteins. Splice sites have been seen to function as cis-acting repressor sequendes (CRS) and inhibit expression of the Rev-dependent RNAs. In order to analyze the role of a splice donor in Rev dependence, the wild-type 5' splice donor of HIV-1 was mutated in the context of other gag sequences. Following transient transfection, RNA expression by RT-PCR was analyzed. The unspliced RNA produced by the mutant construct still required Rev for the cytoplasmic accumulation of the RNA. Despite deletion of the wild-type 5' splice donor and the tat splice acceptor was used. A cryptic splice donor was identified by PCR and subsequent cloning of the spliced RNA. The cryptic site is 5/9 to the consensus sequence and located immediately downstream of the initiation codon (ATG) for Gag. Analysis of the RNA product containing the cryptic splice donor revealed that the Rev was required for the cytoplasmic accumulation of unspliced RNA, while spliced RNA was Rev independent. Transfection of a wild-type construct also demonstrated usage of the cryptic splice donor. These results indicate that a cryptic splice donor can be activated when the wild-type splice donor is inactivated and that the cryptic splice donor may retain Rev regulation. The findings also suggest the potential for cryptic splice sites to serve as CRS in the determining the Rev dependence of viral RNAs.     

Usage of cryptic splice sites in citrullinemia fibroblasts suggests role of polyadenylation in splice-site selection during terminal exon definition

Citrullinemia is a human genetic disease caused by a deficient argininosuccinate synthetase. In fibroblasts established from a citrullinemia patient with a mutation at the 3' splice site of the terminal intron of the gene, three cryptic 3' splice sites; i.e., SA1275, SA1636, and SA1663, residing on the terminal exon were activated. The usage of the cryptic sites showed a gradient, with the most downstream site having the highest usage; i.e., SA1663 > SA1636 > SA1275. However, when these cryptic sites were relocated to the internal exon, SA1636 was used the most. The splice-site strength of SA1636 was at least 10-fold higher than that of SA1663 in this situation. The results suggest that the preferential usage of SA1663 residing on the terminal exon may depend on its proximity to the poly(A) signal rather than on the strength of the splice site. Furthermore, when the strength of the downstream-most splice site increased, almost all the RNAs spliced to this site. However, in the presence of the wild-type splice site, all the RNAs were processed to the authentic site. Apparently, the selection of splice site can be revealed only when the sites being selected do not differ too much in their strength. By using a naturally occurring human mutant gene as a model, this study reveals that polyadenylation may play an important role in the selection of splice site during terminal exon definition.     

Identification of two different mutations causing protein S deficiency in two unrelated Belgian families using a nonisotopic scanning and sequencing method

Hereditary protein S deficiency is a risk factor for developing recurrent venous thromboembolic disease and is caused by a defect in the protein S 1 (PROS1) gene. Identification of the mutation in the PROS1 gene can overcome diagnostic uncertainty in family members with borderline protein S levels. We describe a novel nonisotopic method for molecular diagnosis of protein S deficiency, using fluorescein-labeled amplification and sequencing primers. As a first step, all exons of the PROS1 gene are selectively amplified, and heteroduplex analysis is performed. As a second step, all exons are analyzed by direct sequencing. Using this method, we have characterized the molecular defect in two Belgian families with hereditary protein S deficiency type I: a frameshift mutation in exon XIV (1881insTC) and a missense mutation caused by a T-to-C transition, resulting in substitution of Leu405 by Pro (L405P).     

Splicing defects in the COL3A1 gene: marked preference for 5' (donor) spice-site mutations in patients with exon-skipping mutations and Ehlers-Danlos syndrome type IV

Ehlers-Danlos syndrome (EDS) type IV results from mutations in the COL3A1 gene, which encodes the constituent chains of type III procollagen. We have identified, in 33 unrelated individuals or families with EDS type IV, mutations that affect splicing, of which 30 are point mutations at splice junctions and 3 are small deletions that remove splice-junction sequences and partial exon sequences. Except for one point mutation at a donor site, which leads to partial intron inclusion, and a single base-pair substitution at an acceptor site, which gives rise to inclusion of the complete upstream intron into the mature mRNA, all mutations result in deletion of a single exon as the only splice alteration. Of the exon-skipping mutations that are due to single base substitutions, which we have identified in 28 separate individuals, only two affect the splice-acceptor site. The underrepresentation of splice acceptor-site mutations suggests that the favored consequence of 3' mutations is the use of an alternative acceptor site that creates a null allele with a premature-termination codon. The phenotypes of those mutations may differ, with respect to either their severity or their symptomatic range, from the usual presentation of EDS type IV and thus have been excluded from analysis.     

Splice site prediction in Arabidopsis thaliana pre-mRNA by combining local and global sequence information

Artificial neural networks have been combined with a rule based system to predict intron splice sites in the dicot plant Arabidopsis thaliana. A two step prediction scheme, where a global prediction of the coding potential regulates a cutoff level for a local prediction of splice sites, is refined by rules based on splice site confidence values, prediction scores, coding context and distances between potential splice sites. In this approach, the prediction of splice sites mutually affect each other in a non-local manner. The combined approach drastically reduces the large amount of false positive splice sites normally haunting splice site prediction. An analysis of the errors made by the networks in the first step of the method revealed a previously unknown feature, a frequent T-tract prolongation containing cryptic acceptor sites in the 5' end of exons. The method presented here has been compared with three other approaches, GeneFinder, Gene-Mark and Grail. Overall the method presented here is an order of magnitude better. We show that the new method is able to find a donor site in the coding sequence for the jelly fish Green Fluorescent Protein, exactly at the position that was experimentally observed in A.thaliana transformants. Predictions for alternatively spliced genes are also presented, together with examples of genes from other dicots, monocots and algae. The method has been made available through electronic mail (NetPlantGene@cbs.dtu.dk), or the WWW at http://www.cbs.dtu.dk/NetPlantGene.html     

A T to G mutation in the polypyrimidine tract of the second intron of the human beta-globin gene reduces in vitro splicing efficiency: evidence for an increased hnRNP C interaction

In a patient with a beta-thalassemia intermedia, a mutation was identified in the second intron of the human beta-globin gene. The U-->G mutation is located within the polypyrimidine tract at position -8 upstream of the 3' splice site. In vivo, this mutation leads to decreased levels of the hemoglobin protein. Because of the location of the mutation and the role of the polypyrimidine tract in the splicing process, we performed in vitro splicing assays on the pre-messenger RNA (pre-mRNA). We found that the splicing efficiency of the mutant pre-mRNA is reduced compared to the wild type and that no cryptic splice sites are activated. Analysis of splicing complex formation shows that the U-->G mutation affects predominantly the progression of the H complex towards the pre-spliceosome complex. By cross-linking and immunoprecipitation assays, we show that the hnRNP C protein interacts more efficiently with the mutant precursor than with the wild-type. This stronger interaction could play a role, directly or indirectly, in the decreased splicing efficiency.     

Study on the elimination of Angiostrongylus costaricensis first stage larvae in the experimental infection of Swiss mice

We report six of 16 U.K. melanoma families and two of 17 patients with multiple primary melanomas and a negative family history who have between them four different functionally damaging mutations of the CDKN2A (p16) gene: an Arg 24 Pro substitution in exon 1 in one family, a stop codon at codon 44 of exon 1 in one family, and a Met 53 Ile substitution in exon 2 in four families. One multiple primary melanoma patient also has the Met 53 Ile mutation and a second has a G-T substitution at the IVS2 + 1 splice donor site. Our data together with other recent publications from France and the U.S.A. indicate that screening melanoma kindreds with only two affected family members for CDKN2A mutations is justified.     

Splice-site mutations: a novel genetic mechanism of Crigler-Najjar syndrome type 1

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.     

Detection of postoperative myocardial ischemia by bedside ST-segment analysis in coronary artery bypass graft patients

INTRODUCTION: Inherited long QT syndrome (LQTS) recently has been associated with mutations in genes coding for potassium (KVLQT1, KCNE1, and HERG) or sodium (SCN5A) ion channels involved in regulating either sodium inward or potassium outward currents of heart cells, resulting in prolongation of the repolarization period. We describe a new mutation, a -1 donor splice site mutation in a kindred with two affected members (QTc = 0.61 and 0.54 sec). METHODS AND RESULTS: Single stranded conformation polymorphism (SSCP) analyses were performed on DNA fragments amplified by polymerase chain reaction from DNA extracted from whole blood. Aberrant conformers were analyzed by DNA sequencing. SSCP analysis of the KVLQT1 gene revealed an aberrant conformer in the affected family members. DNA sequencing confirmed the presence of a G-->A change in the last nucleotide of codon 344. This mutation does not cause an amino acid change, but a change of the splice site characteristics at the 3' end of exon 6. The mutation may affect, through deficient splicing, the putative sixth transmembrane segment of the K+ channel, and this type of mutation has not previously been described in KVLQT1. CONCLUSION: The clinical course of LQTS in the affected family members, in whom no deaths occurred despite 20 to 30 syncopes, can be explained by the ability of the cellular machinery to perform partial correct splicing in the mutant allele. This type of mutation may be misinterpreted as a normal variant, since it is a point mutation causing neither an amino acid change nor the introduction of a stop codon.     

Circulating endotoxin during initial antibiotic treatment of severe gram-negative bacteremic infections

The second most common cause of congenital adrenal hyperplasia is 11 beta-hydroxylase deficiency, an autosomal recessive disorder. We performed genetic analysis of CYP11B1, the gene encoding steroid 11 beta-hydroxylase, in three patients with classic 11 beta-hydroxylase deficiency. Herein we describe the first splice donor site mutation, a new nonsense mutation, and a new missense mutation in this disorder. An African-American patient was found to be a compound heterozygote for a codon 318 + 1G --> A substitution at the 5'-splice donor site of intron 5, in combination with Q356X, a nonsense mutation previously reported in an African-American patient. A Caucasian patient was found to be a compound heterozygote with a novel missense mutation, T318R, in combination with a previously reported 28-bp deletion in exon 2. A different mutation at codon 318 (T318M) has been described previously. A Caucasian patient was heterozygous for a novel nonsense mutation (Q19X) in exon 2. The second mutation was not identified in this patient. Multiple apparent polymorphisms were also observed. Two of these polymorphisms in CYP11B1 represent sequences from CYP11B2, suggesting that gene conversion may have occurred. In summary, we have identified three novel mutations and two previously reported mutations in CYP11B1 patients with 11 beta-hydroxylase deficiency. Our data suggest the presence of a mutational hot spot at codon 318 of CYP11B1, and the possibility of a founder effect in frequently identified mutations.