The Missense p.S231F Phenylalanine Hydroxylase Gene Mutation Causes Complete Loss of Enzymatic Activity In Vitro

Phenylketonuria (PKU), the most frequent disorder of amino acid metabolism, is caused by mutations in human phenylalanine hydroxylase gene (PAH), leading to deficient enzyme activity. Previously reported but uncharacterized PAH gene mutation, p.S231F (c.692C > T), was detected in Serbian patients with classical PKU. We analyzed p.S231F PAH protein in prokaryotic (Escherichia coli) and eukaryotic expression system (hepatoma cells). In both systems the mutant enzyme was unstable. Residual enzyme activity in vitro was ~1%. Mutation p.S231F PAH was not activated by pre-incubation with phenylalanine substrate. We found no GroEL/GroES chaperone effect and slightly positive effect of the (6R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH₄) on the stabilization of the protein structure. Our findings were in accordance with severe patients’ phenotypes. In conclusion, p.S231F should be classified as a functionally null PAH gene mutation as it drastically reduces stability and activity of the PAH enzyme in vitro.     

Activation of cryptic splice sites is a frequent splicing defect mechanism caused by mutations in exon and intron sequences of the OPA1 gene

Mutations in OPA1 are the most frequent cause underlying autosomal dominant optic atrophy (adOA). Until now only few putative splicing mutations in the OPA1 gene have been investigated at the mRNA level and all these result in exon skipping. Here, we report the identification and cDNA analysis of four intronic and three exonic OPA1 gene mutations that cause a variety of splicing defects including activation of cryptic splice sites in either flanking exon or intron sequences, and a leaky splicing mutation. Our results show that cDNA analysis is of prime importance for the full evaluation of the effect of putative splicing mutations in the OPA1 gene.     

Predicting a clinical/biochemical phenotype for PKU/MHP patients with PAH gene mutations

Phenylketonuria (PKU) and mild hyperphenylalaninemia (MHP) are allelic disorders caused by mutations in the gene encoding phenylalanine hydroxylase (PAH). In this study, a total of 218 independent PAH chromosomes (109 unrelated patients with PKU residing in Lithuania) were investigated. All 13 exons of the PAH gene of all PKU probands were scanned for DNA alterations by denaturing gradient gel electrophoresis (DGGE). In the cases of a specific DGGE pattern recognized, mutations were identified by direct fluorescent automated sequencing or by restriction enzyme digestion analysis of relevant exons. Twenty-five different PAH gene mutations were identified in Lithuania. We estimated a connection between individual PAH locus mutations and biochemical and metabolic phenotypes in patients in whom the mutant allele acts on its own, i.e., in functionally hemizygous patients and using the assigned value (AV) method to determine the severity of both common and rare mutant alleles, as well as to check a model to predict the combined phenotypic effect of two mutant PAH alleles. The text was submitted authors English.     

Autoregulation of avian renal plasma flow: contribution of the renal portal system

Summary A simplified avian kidney model was used to assess renal plasma flow (RPF) at normal (100–110 mmHg) or unilaterally reduced (40–50 mmHg) renal arterial perfusion pressure (RAPP) in domestic fowl with ambient (AMBIENT group) or restricted (RESTRICTED group) renal portal flow. Direct measurement of para-aminohippuric acid (PAH) extraction efficiencies (E_PAH) allowed avian RPF to be calculated from the clearance of PAH (C_PAH). E_PAH was unaffected by RAPP, thereby validating the use of PAH to estimate RPF during experimental hemodynamic manipulations. C_PAH and RPF were unaffected by RAPP in the AMBIENT group (perfect autoregulation), but decreased significantly compared with contralateral kidney values during reduction of RAPP in the RESTRICTED group. Urine flow and glomerular filtration rates (GFR) were reduced unilaterally along with RAPP, regardless of the portal perfusion status. The renal portal system contributes to overall RPF autoregulation in domestic fowl, helping to maintain constancy of renal blood flow even after RAPP is reduced well below the GFR autoregulatory limit.Abbreviations BW body weight - C _In Clearance of inulin - C _PAH clearance of PAH - E _PAH PAH extraction efficiency - FF filtration fraction - GFR glomerular filtration rate - LiOH lithium hydroxide - MT mammalian-type nephron - PAH para-aminohippuric acid - [PAH] _A concentration of PAH in arterial plasma - [PAH] _a chromagen corrected PAH in arterial plasma - [PAH] _E endogenous PAH-like chromagen - [PAH] _UF concentration of ultrafilterable PAH - [PAH] _v concentration of PAH in renal venous plasma - [PAH] _v chromagen corrected PAH in renal venous plasma - RAPP renal arterial perfusion pressure - RPF renal plasma flow - RT reptilian-type nephron - UFR urine flow rate - UFR per gram urine flow rate per gram kidney weight - T _M S PAH tubular secretory maximum for PAH - SEM standard error of mean     

Functional Analysis of Mutations in Exon 9 of NF1 Reveals the Presence of Several Elements Regulating Splicing

Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.     

In vivo analysis of intron processing using splicing-dependent reporter gene assays

The mechanisms of intron recognition and processing have been well-studied in mammals and yeast, but in plants the biochemistry of splicing is not known and the rules for intron recognition are not clearly defined. To increase understanding of intron processing in plants, we have constructed new pairs of vectors, pSuccess and pFail, to assess the efficiency of splicing in maize cultured cells. In the pFail series we use translation of pre-mRNA to monitor the amount of unspliced RNA. We inserted an ATG codon in the Bz2 (Bronze-2) intron in frame with luciferase: this construct will express luciferase activity only when splicing fails. In the pSuccess series the spliced message is monitored by inserting an ATG upstream of the Bz2 intron in frame with luciferase: this construct will express luciferase activity only when splicing succeeds. We show here, using both the wild-type Bz2 intron and the same intron with splice site mutations, that the efficiency of splicing can be estimated by the ratio between the luciferase activities of the vector pairs. We also show that mutations in the unique U-rich motif inside the intron can modulate splicing. In addition, a GC-rich insertion in the first exon increases the efficiency of splicing, suggesting that exons also play an important role in intron recognition and/or processing.     

The differential expression of alternatively spliced transcripts and imprinting status of MEG9 gene in cows

Meg9/Mirg (maternally expressed gene 9/microRNA containing gene), a non‐coding RNA (ncRNA) comprising many alternatively splicing isoforms, has been identified as maternally expressed in mouse and sheep, but its imprinting status and splicing variants are still unknown in cattle. In this study, we found three splicing variants of the cattle MEG9 gene expressed in a tissue‐specific manner. A single nucleotide polymorphism site (SNP c.1354C>G) was identified in exon 3 of cattle MEG9 and used to distinguish between monoallelic and biallelic expression. Our results showed that MEG9 exhibited monoallelic expression in all examined cattle tissues by comparing sequencing results between genomic DNA and cDNA levels at the c.1354C>G SNP site, suggesting that MEG9 is imprinted in cattle.     

Genotyping of patients with phenylketonuria from different regions of Russia for determining BH4 responsiveness

To date, the efficacy of the phenylalanine hydroxylase (PAH) cofactor is proved for the treatment of both BH4-dependent hyperphenylalaninemia and phenylketonuria patients with mutations in the PAH gene. Since the patient’s response depends on the presence of residual PAH enzyme activity, it is advisable to search for mutations in the PAH gene to identify the potential responders and nonresponders to therapy. Four hundred thirty-five phenylketonuria patients from 13 regions of the Russian Federation were genotyped in order to identify responders and nonresponders to tetrahydrobiopterin (BH4) therapy. According to the results of this study, the number of probable nonresponders to the BH4 treatment exceeds 50% owing to a higher overall allelic frequency of “severe” PAH gene mutations. Responder patients with two “mild” mutations in the PAH gene were identified (1.6%).     

Characterization of Mutations at the Mouse Phenylalanine Hydroxylase Locus

Two genetic mouse models for human phenylketonuria have been characterized by DNA sequence analysis. For each, a distinct mutation was identified within the protein coding sequence of the phenylalanine hydroxylase gene. This establishes that the mutated locus is the same as that causing human phenylketonuria and allows a comparison between these mouse phenylketonuria models and the human disease. A genotype/phenotype relationship that is strikingly similar to the human disease emerges, underscoring the similarity of phenylketonuria in mouse and man. InPAH^ENU1,the phenotype is mild. ThePah^enu1mutation predicts a conservative valine to alanine amino acid substitution and is located in exon 3, a gene region where serious mutations are rare in humans. InPAH^ENU2,the phenotype is severe. ThePah^enu2mutation predicts a radical phenylalanine to serine substitution and is located in exon 7, a gene region where serious mutations are common in humans. InPAH^ENU2,the sequence information was used to devise a direct genotyping system based on the creation of a newAlw26I restriction endonuclease site.     

Evidence of functional selection pressure for alternative splicingevents that accelerate evolution of protein subsequences

Recently, it was proposed that alternative splicing may act as a mechanism for opening accelerated paths of evolution, by reducing negative selection pressure, but there has been little evidence so far whether this could produce adaptive benefit. Here we employ metrics of very different types of selection pressures (e.g. against amino acid mutations (Ka/Ks); against mutations at synonymous sites (Ks); and for protein reading-frame preservation) to address this question via genome-wide analyses of human, chimpanzee, mouse, and rat. These data show that alternative splicing relaxes Ka/Ks selection pressure up to seven-fold, but intriguingly that this effect is accompanied by a strong increase in selection pressure against synonymous mutations, which propagates into the adjacent intron, and correlates strongly with the alternative splicing level observed for each exon. These effects are highly local to the alternatively spliced exon. Comparisons of these four genomes consistently show an increase in the density of amino acid mutations (Ka) in alternatively spliced exons, and a decrease in the density of synonymous mutations (Ks). This selection pressure against synonymous mutations in alternatively spliced exons was accompanied in all four genomes by a striking increase in selection pressure for protein reading-frame preservation, and both increased markedly with increasing evolutionary age. Restricting our analysis to a subset of exons with strong evidence for biologically functional alternative splicing produced identical results. Thus alternative splicing apparently can create evolutionary “hotspots” within a protein sequence, and these events have evidently been selected for during mammalian evolution.     

The mechanism of sex-specific splicing at the doublesex gene is different between Drosophila melanogaster and Bombyx mori

We have previously reported that Bmdsx, a homologue of the sex-determining gene, doublesex (dsx), was found to be sex-specifically expressed in various tissues at larval, pupal, and adult stages in the silkworm, Bombyx mori, and was alternatively spliced to yield male- and female-specific mRNAs. To reveal sex-specific differences in splicing patterns of Bmdsx pre-mRNA, the genomic sequence was determined and compared with male- and female-specific Bmdsx cDNA sequences. The open reading frame (ORF) consisted of five exons. Exons 3 and 4 were specifically incorporated into the female type of Bmdsx mRNA. On the other hand, exon 2 was spliced to exon 5 to produce the male type mRNA of Bmdsx. As in the case of Drosophila dsx, the OD2 domain was separated by a female-specific intron into sex-independent and sex-dependent regions. Sex-specific splicing occurred in equivalent positions in the Drosophila dsx gene. However, unlike Drosophila dsx, the female-specific introns showed no weak 3′ splice sites, and the TRA/TRA-2 binding site related sequences were not found in the female-specific exon, nor even in any other regions of the Bmdsx gene. Moreover, an in vitro splicing reaction consisting of HeLa cell nuclear extracts showed that the female-type of Bmdsx mRNA represented the default splicing. These findings suggest that the structural features of the sex-specific splicing patterns of Bmdsx pre-mRNA are similar to those of Drosophila dsx but the regulation of sex-specific alternative splicing of Bmdsx pre-mRNA is different.     

Rbfox-regulated alternative splicing is critical for zebrafish cardiac and skeletal muscle functions

Rbfox RNA binding proteins are implicated as regulators of phylogenetically-conserved alternative splicing events important for muscle function. To investigate the function of rbfox genes, we used morpholino-mediated knockdown of muscle-expressed rbfox1l and rbfox2 in zebrafish embryos. Single and double morphant embryos exhibited changes in splicing of overlapping sets of bioinformatically-predicted rbfox target exons, many of which exhibit a muscle-enriched splicing pattern that is conserved in vertebrates. Thus, conservation of intronic Rbfox binding motifs is a good predictor of Rbfox-regulated alternative splicing. Morphology and development of single morphant embryos were strikingly normal; however, muscle development in double morphants was severely disrupted. Defects in cardiac muscle were marked by reduced heart rate and in skeletal muscle by complete paralysis. The predominance of wavy myofibers and abnormal thick and thin filaments in skeletal muscle revealed that myofibril assembly is defective and disorganized in double morphants. Ultra-structural analysis revealed that although sarcomeres with electron dense M- and Z-bands are present in muscle fibers of rbfox1l/rbox2 morphants, they are substantially reduced in number and alignment. Importantly, splicing changes and morphological defects were rescued by expression of morpholino-resistant rbfox cDNA. Additionally, a target-blocking MO complementary to a single UGCAUG motif adjacent to an rbfox target exon of fxr1 inhibited inclusion in a similar manner to rbfox knockdown, providing evidence that Rbfox regulates the splicing of target exons via direct binding to intronic regulatory motifs. We conclude that Rbfox proteins regulate an alternative splicing program essential for vertebrate heart and skeletal muscle functions.     

Seemingly neutral polymorphic variants may confer immunity to splicing-inactivating mutations: a synonymous SNP in exon 5 of MCAD protects from deleterious mutations in a flanking exonic splicing enhancer

The idea that point mutations in exons may affect splicing is intriguing and adds an additional layer of complexity when evaluating their possible effects. Even in the best-studied examples, the molecular mechanisms are not fully understood. Here, we use patient cells, model minigenes, and in vitro assays to show that a missense mutation in exon 5 of the medium-chain acyl-CoA dehydrogenase (MCAD) gene primarily causes exon skipping by inactivating a crucial exonic splicing enhancer (ESE), thus leading to loss of a functional protein and to MCAD deficiency. This ESE functions by antagonizing a juxtaposed exonic splicing silencer (ESS) and is necessary to define a suboptimal 3′ splice site. Remarkably, a synonymous polymorphic variation in MCAD exon 5 inactivates the ESS, and, although this has no effect on splicing by itself, it makes splicing immune to deleterious mutations in the ESE. Furthermore, the region of MCAD exon 5 that harbors these elements is nearly identical to the exon 7 region of the survival of motor neuron (SMN) genes that contains the deleterious silent mutation in SMN2, indicating a very similar and finely tuned interplay between regulatory elements in these two genes. Our findings illustrate a mechanism for dramatic context-dependent effects of single-nucleotide polymorphisms on gene-expression regulation and show that it is essential that potential deleterious effects of mutations on splicing be evaluated in the context of the relevant haplotype.     

Mutations in argininosuccinate synthetase mRNA of Japanese patients,causing classical citrullinemia.

Citrullinemia is an autosomal recessive disease caused by a genetic deficiency of argininosuccinate synthetase. In order to characterize mutations in Japanese patients with classical citrullinemia, RNA isolated from 10 unrelated patients was reverse-transcribed, and cDNA amplified by PCR was cloned and sequenced. The 10 mutations identified included 6 missense mutations (A118T, A192V, R272C, G280R, R304W, and R363L), 2 mutations associated with an absence of an exon 7 or exon 13, 1 mutation with a deletion of the first 7 bp in exon 16 (which might be caused by abnormal splicing), and 1 mutation with an insertion of 37 bp within exons 15 and 16 in cDNA. The insertion mutation and the five missense mutations (R304W being excluded) are new mutations described in the present paper. These are in addition to 14 mutations (9 missense mutations, 4 mutations associated with an absence of an exon in mRNA, and 1 splicing mutation) that we identified previously in mainly American patients with neonatal citrullinemia. Two of these 20 mutations, a deletion of exon 13 sequence and a 7-bp deletion in exon 16, were common to Japanese and American populations from different ethnic backgrounds; however, other mutations were unique to each population. Furthermore, the presence of a frequent mutation--the exon 7 deletion mutation in mRNA, which accounts for 10 of 23 affected alleles--was demonstrated in Japanese citrullinemia. This differs from the situation in the United States, where there was far greater heterogeneity of mutations.     

FBN1 isoform expression varies in a tissue and development-specific fashion

Mutations in FBN1 cause Marfan syndrome, a heritable disorder of connective tissue. FBN1 encodes the extracellular matrix protein, fibrillin. Our objective was to elucidate the extent that variation in RNA splicing contributes to FBN1 isoforms. To identify FBN1 splice variants, we scanned each of its 64 internal exons in a set of pooled human brain cDNA samples. FBN1 splicing is generally efficient as we identified only two variants. Neither variant has previously been reported in the literature and include (i) an isoform which contains a cryptic 105 basepair exon between exons 54 and 55 (54A-FBN1) and (ii) an isoform which contains a cryptic 62 basepair exon between exons 57 and 58 (57A-FBN1). We compared 57A-FBN1 and FBN1 expression in multiple human tissues, including adult skeletal muscle and brain, as well as fetal skeletal muscle, brain, liver, aorta, lung, skin, and heart. 57A-FBN1 represents 8–44% of FBN1 mRNA and varies in a tissue- and development-specific fashion. In adult brain, 57A-FBN1 represented 39 ± 3 (%, mean ± SD) of total FBN1 expression. In contrast, 57A-FBN1 represented 19 ± 2 (%, mean ± SD) of FBN1 expression in skeletal muscle. In fetal tissue, the 57A-FBN1 proportion was highest in brain (27%) and low elsewhere, e.g., skin, aorta and lung (9–13%). In summary, a significant proportion of FBN1 is expressed as 57A-FBN1 and this proportion varies in a tissue- and development-specific fashion. Since the 57A insertion creates a premature stop codon that mimics Marfan-associated mutations, the protein encoded by 57A-FBN1 is likely to not be functional. These results suggest that altered splicing may modulate disease severity, regulate FBN1 expression, and potentially represent a therapeutic target.     

GT to AT transition at a splice donor site causes skipping of the preceding exon in phenylketonuria.

Classical Phenylketonuria (PKU) is an autosomal recessive human genetic disorder caused by a deficiency of hepatic phenylalanine hydroxylase (PAH). We isolated several mutant PAH cDNA clones from a PKU carrier individual and showed that they contained an internal 116 base pair deletion, corresponding precisely to exon 12 of the human chromosomal PAH gene. The deletion causes the synthesis of a truncated protein lacking the C-terminal 52 amino acids. Gene transfer and expression studies using the mutant PAH cDNA indicated that the deletion abolishes PAH activity in the cell as a result of protein instability. To determine the molecular basis of the deletion, the mutant chromosomal PAH gene was isolated from this individual and shown to contain a GT-- greater than AT substitution at the 5' splice donor site of intron 12. Thus, the consequence of the splice donor site mutation in the human liver is the skipping of the preceding exon during RNA splicing.     

Expression of cDNAs encoding mouse cardiac troponin T isoforms: characterization of a large sample of independent clones

We have isolated 52 mouse cardiac troponin-T-encoding cDNA clones (TnT) by specific antibody screening of a λZAPII expression library. Sequencing data from the large sample of independent cDNAs demonstrated relationships among the expression of four alternatively-spliced exons of the cardiac TnT gene, producing seven classes of cDNAs encoding four protein isoforms differing in two variable regions. In the N-terminal variable region and next to the embryonic-specific exon 4, an alternatively spliced exon 3a was identified in 20% of the adult isoforms. The alternatively spliced exon 12, corresponding to a central variable region between the two functional domains of TnT, was found in approx. 79% of the 52 mouse cardiac TnT cDNAs with a single base mutation completely abolishing the splicing at an internal acceptor site. Three novel alternative splicing acceptor sites in the 5'-untranslated portion of exon 2 have been identified with different frequencies.     

Effect of plant growth promoting bacterium; Pseudomonas putida UW4 inoculation on phytoremediation efficacy of monoculture and mixed culture of selected plant species for PAH and lead spiked soils

Plant growth promoting bacteria (PGPB) enhanced phytoremediation (PEP) is an attractive remedial strategy for the remediation of polycyclic aromatic hydrocarbon (PAH) and heavy metal (HM) contaminated sites. The effect of PGPB; Pseudomonas putida UW4 inoculation on the phytoremediation efficiency of Medicago sativa, Festuca arundinacea, Lolium perenne, and mixed plants (L. perenne and F. arundinacea) was assessed. This involved two contaminant treatments; “PAH” (phenanthrene; 300?mg·kg^?1, fluoranthene; 200?mg·kg^?1, and benzo[a]pyrene; 5?mg·kg^?1) and “PAH?+?HM” (‘PAH’ treatments +100?mg of Pb/kg). PGPB inoculation significantly enhanced root biomass yield of F. arundinacea in PAH treatment, and the mixed plant shoot biomass and L. perenne root biomass yields of the PAH?+?HM treatment. PGPB significantly enhanced dissipation of phenanthrene and fluoranthene for M. sativa-PAH?+?PGPB treatment and fluoranthene for F. arundinacea-PAH?+?HM?+?PGPB treatment. In others, PGPB inoculation either had no impact or inhibited PAH dissipation. PAH dissipation for the single and mixed plant treatments with PGPB inoculation were not different. The efficiency of PEP is dependent on different factors such as PGPB inoculum biomass, plant species, plant–microbe specificity and type of contaminants. Exploiting PEP technology would require proper understanding of plant tolerance and growth promoting mechanisms, and rhizosphere activities.