CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.

Standard CRISPR-mediated gene disruption strategies rely on Cas9-induced DNA double-strand breaks (DSBs). Here, we show that CRISPR-dependent base editing efficiently inactivates genes by precisely converting four codons (CAA, CAG, CGA, and TGG) into STOP codons without DSB formation. To facilitate gene inactivation by induction of STOP codons (iSTOP), we provide access to a database of over 3.4 million single guide RNAs (sgRNAs) for iSTOP (sgSTOPs) targeting 97%-99% of genes in eight eukaryotic species, and we describe a restriction fragment length polymorphism (RFLP) assay that allows the rapid detection of iSTOP-mediated editing in cell populations and clones. To simplify the selection of sgSTOPs, our resource includes annotations for off-target propensity, percentage of isoforms targeted, prediction of nonsense-mediated decay, and restriction enzymes for RFLP analysis. Additionally, our database includes sgSTOPs that could be employed to precisely model over 32,000 cancer-associated nonsense mutations. Altogether, this work provides a comprehensive resource for DSB-free gene disruption by iSTOP.

Variation in Release Factor Abundance Is Not Needed to Explain Trends in Bacterial Stop Codon Usage.

In bacteria stop codons are recognized by one of two class I release factors (RF1) recognizing TAG, RF2 recognizing TGA, and TAA being recognized by both. Variation across bacteria in the relative abundance of RF1 and RF2 is thus hypothesized to select for different TGA/TAG usage. This has been supported by correlations between TAG:TGA ratios and RF1:RF2 ratios across multiple bacterial species, potentially also explaining why TAG usage is approximately constant despite extensive variation in GC content. It is, however, possible that stop codon trends are determined by other forces and that RF ratios adapt to stop codon usage, rather than vice versa. Here, we determine which direction of the causal arrow is the more parsimonious. Our results support the notion that RF1/RF2 ratios become adapted to stop codon usage as the same trends, notably the anomalous TAG behavior, are seen in contexts where RF1:RF2 ratios cannot be, or are unlikely to be, causative, that is, at 3'untranslated sites never used for translation termination, in intragenomic analyses, and across archaeal species (that possess only one RF1). We conclude that specifics of RF biology are unlikely to fully explain TGA/TAG relative usage. We discuss why the causal relationships for the evolution of synonymous stop codon usage might be different from those affecting synonymous sense codon usage, noting that transitions between TGA and TAG require two-point mutations one of which is likely to be deleterious.

Translation termination in human mitochondria - substrate specificity of mitochondrial release factors.

Mitochondria are the essential players in eukaryotic ATP production by oxidative phosphorylation, which relies on the maintenance and accurate expression of the mitochondrial genome. Even though the basic principles of translation are conserved due to the descendance from a bacterial ancestor, some deviations regarding translation factors as well as mRNA characteristics and the applied genetic code are present in human mitochondria. Together, these features are certain challenges during translation the mitochondrion has to handle. Here, we discuss the current knowledge regarding mitochondrial translation focusing on the termination process and the associated quality control mechanisms. We describe how mtRF1a resembles bacterial RF1 mechanistically and summarize in vitro and recent in vivo data leading to the conclusion of mtRF1a being the major mitochondrial release factor. On the other hand, we discuss the ongoing debate about the function of the second codon-dependent mitochondrial release factor mtRF1 regarding its role as a specialized termination factor. Finally, we link defects in mitochondrial translation termination to the activation of mitochondrial rescue mechanisms highlighting the importance of ribosome-associated quality control for sufficient respiratory function and therefore for human health.

Factors Affecting Readthrough of Natural Versus Premature Termination Codons.

Nonsense mutations occur within the open-reading frame of a gene resulting in a premature termination codon (PTC). PTC-containing mRNAs can either be degeraded or cause premature translation termination producing a truncated protein that can be either nonfunctional or toxic. Translational readthrough inducing drugs (TRIDs) are small molecules that are able to induce readthrough, resulting in the restoration of full-length protein expression. The re-expressed proteins usually harbor a missense change. The effciency of individual TRIDs is variable and varies between different genes and even different nonsense mutations in the same gene. This review summarizes factors, including the sequences located upstream and downstream the disease-causing mutation and the type of PTC, affecting the translational readthrough process by modulating the type of amino acid insertion and the efficiency of the process during readthrough following TRIDs treatments.

Effective Population Size Predicts Local Rates but Not Local Mitigation of Read-through Errors.

In correctly predicting that selection efficiency is positively correlated with the effective population size (Ne), the nearly neutral theory provides a coherent understanding of between-species variation in numerous genomic parameters, including heritable error (germline mutation) rates. Does the same theory also explain variation in phenotypic error rates and in abundance of error mitigation mechanisms? Translational read-through provides a model to investigate both issues as it is common, mostly nonadaptive, and has good proxy for rate (TAA being the least leaky stop codon) and potential error mitigation via "fail-safe" 3' additional stop codons (ASCs). Prior theory of translational read-through has suggested that when population sizes are high, weak selection for local mitigation can be effective thus predicting a positive correlation between ASC enrichment and Ne. Contra to prediction, we find that ASC enrichment is not correlated with Ne. ASC enrichment, although highly phylogenetically patchy, is, however, more common both in unicellular species and in genes expressed in unicellular modes in multicellular species. By contrast, Ne does positively correlate with TAA enrichment. These results imply that local phenotypic error rates, not local mitigation rates, are consistent with a drift barrier/nearly neutral model.

CRISPR-BETS: a base-editing design tool for generating stop codons.

Cytosine base editors (CBEs) can install a predefined stop codon at the target site, representing a more predictable and neater method for creating genetic knockouts without altering the genome size. Due to the enhanced predictability of the editing outcomes, it is also more efficient to obtain homozygous mutants in the first generation. With the recent advancement of CBEs on improved editing activity, purify and specificity in plants and animals, base editing has become a more appealing technology for generating knockouts. However, there is a lack of design tools that can aid the adoption of CBEs for achieving such a purpose, especially in plants. Here, we developed a user-friendly design tool named CRISPR-BETS (base editing to stop), which helps with guide RNA (gRNA) design for introducing stop codons in the protein-coding genes of interest. We demonstrated in rice and tomato that CRISPR-BETS is easy-to-use, and its generated gRNAs are highly specific and efficient for generating stop codons and obtaining homozygous knockout lines. While we tailored the tool for the plant research community, CRISPR-BETS can also serve non-plant species.

A Respiratory Syncytial Virus Attachment Gene Variant Associated with More Severe Disease in Infants Decreases Fusion Protein Expression, Which May Facilitate Immune Evasion.

This study identified a genotype of respiratory syncytial virus (RSV) associated with increased acute respiratory disease severity in a cohort of previously healthy term infants. The genotype (2stop+A4G) consists of two components. The A4G component is a prevalent point mutation in the 4th position of the gene end transcription termination signal of the G gene of currently circulating RSV strains. The 2stop component is two tandem stop codons at the G gene terminus, preceding the gene end transcription termination signal. To investigate the biological role of these RSV G gene mutations, recombinant RSV strains harboring either a wild-type A2 strain G gene (one stop codon preceding a wild-type gene end signal), an A4G gene end signal preceded by one stop codon, or the 2stop+A4G virulence-associated combination were generated and characterized. Infection with the recombinant A4G (rA4G) RSV mutant resulted in transcriptional readthrough and lower G and fusion (F) protein levels than for the wild type. Addition of a second stop codon preceding the A4G point mutation (2stop+A4G) restored G protein expression but retained lower F protein levels. These data suggest that RSV G and F glycoprotein expression is regulated by transcriptional and translational readthrough. Notably, while rA4G and r2stop+A4G RSV were attenuated in cells and in naive BALB/c mice compared to that for wild-type RSV, the r2stop+A4G RSV was better able to infect BALB/c mice in the presence of preexisting immunity than rA4G RSV. Together, these factors may contribute to the maintenance and virulence of the 2stop+A4G genotype in currently circulating RSV-A strains.IMPORTANCE Strain-specific differences in respiratory syncytial virus (RSV) isolates are associated with differential pathogenesis in mice. However, the role of RSV genotypes in human infection is incompletely understood. This work demonstrates that one such genotype, 2stop+A4G, present in the RSV attachment (G) gene terminus is associated with greater infant disease severity. The genotype consists of two tandem stop codons preceding an A-to-G point mutation in the 4th position of the G gene end transcription termination signal. Virologically, the 2stop+A4G RSV genotype results in reduced levels of the RSV fusion (F) glycoprotein. A recombinant 2stop+A4G RSV was better able to establish infection in the presence of existing RSV immunity than a virus harboring the common A4G mutation. These data suggest that regulation of G and F expression has implications for virulence and, potentially, immune evasion.

Assessment of Internalin A Gene Sequences and Cell Adhesion and Invasion Capacity of Listeria monocytogenes Strains Isolated from Foods of Animal and Related Origins.

Listeria monocytogenes is a foodborne pathogen of global relevance that causes outbreaks and sporadic cases of listeriosis, acquired through the consumption of contaminated products, including milk or meat products and ready-to-eat meat products subjected to intensive handling. The objective of the present study was to classify L. monocytogenes isolated from various food-related sources in the Federal District of Brazil and surrounding areas to sequence internalin A (inlA) genes from these isolates and assess their adhesion and invasion capacity using Caco-2 cells. In addition, 15 were classified as group I, 3 as group II, and 7 classified as group IV. Premature stop codons (PMSCs) at the nucleotide position 976 (GAA→TAA) of the inlA gene were identified in 5 of the 25 isolates. Adhesion and invasion tests in Caco-2 cells showed that all the isolates were capable of adhesion and cellular invasion, with isolates containing PMSCs exhibiting on average higher invasion capacity than those without PMSCs (p = 0.041) and a median of adhesion very distinctive from those without stop codons. These results are the first report of PMSCs in the inlA gene of L. monocytogenes from the Federal District of Brazil and Brazil.

Genetic characteristics and virulence of Listeria monocytogenes isolated from fresh vegetables in China.

BACKGROUND: Ready-to-eat (RTE) vegetables have become increasingly popular along with the trend of moving towards a healthy lifestyle. However, RTE vegetables are at a higher risk of containing pathogens, maybe owing to lack of rigorous sanitization procedures. To understand the prevalence and potential risk of Listeria monocytogenes in RTE vegetables, we investigated the contamination level and characteristics of L. monocytogenes isolated from fresh vegetables. RESULTS: Twenty-three (5.49%) of the 419 vegetables samples were positive for L. monocytogenes. Phylogenetic group I.1 (1/2a-3a) and II.2 (1/2b-3b-7) strains were predominant in 30 isolates, which accounted for 33.3 and 50.0%, respectively. Multilocus sequence typing of the 30 isolates grouped them into nine sequence types (STs). The most common STs were ST87 (36.7%) and ST8 (26.7%). Virulence analysis showed that all 30 isolates harbored eight classical virulence genes, 10.0% isolates harbored the llsX gene (ST3 and ST1 strains), and 36.7% carried the ptsA gene and belonged to ST87. Approximately 83.3% isolates carried full-length inlA, whereas five isolates had premature stop codons in inlA, three of which belonged to ST9 and two to ST8. Antibiotic susceptibility showed the isolates were varyingly resistant to 13 antibiotics, 26.7% of the isolates were multi-drug resistant. CONCLUSIONS: The fresh vegetables contain some potential hypervirulent L. monocytogenes (ST1 and ST87) in the Chinese markets. In addition, the high rate of L. monocytogenes isolates was multi-drug resistant. Fresh raw vegetables may be a possible transmission route for L. monocytogenes infection in consumers. Therefore, sanitization of raw fresh vegetables should be strengthened to ensure their microbiological safety when used as RTE vegetables.

Routine drug resistance testing in proviral HIV-1 DNA: Prevalence of stop codons and hypermutation, and associated factors.

We investigated the presence of stop codons (SC) and/or hypermutation (HM) in HIV-1 DNA sequences generated for routine drug resistance testing in proviral HIV-1 DNA, and sought for associated factors. At least one SC was identified in 6.2% of HIV-1 DNA sequences, among which 54.8% were hypermutated. The defective virus group (SC w/o HM) was similar to the non-SC group regarding the characteristics of HIV-1 infection, and before drug exposure. In addition, the HIV-1 DNA levels were not different between both groups. Sequences with SC/HM displayed a higher proportion of RAMs. The impact of the SC/HM associated RAMs on clinical responses requires further investigation.

Immune-escape mutations and stop-codons in HBsAg develop in a large proportion of patients with chronic HBV infection exposed to anti-HBV drugs in Europe.

BACKGROUND: HBsAg immune-escape mutations can favor HBV-transmission also in vaccinated individuals, promote immunosuppression-driven HBV-reactivation, and increase fitness of drug-resistant strains. Stop-codons can enhance HBV oncogenic-properties. Furthermore, as a consequence of the overlapping structure of HBV genome, some immune-escape mutations or stop-codons in HBsAg can derive from drug-resistance mutations in RT. This study is aimed at gaining insight in prevalence and characteristics of immune-associated escape mutations, and stop-codons in HBsAg in chronically HBV-infected patients experiencing nucleos(t)ide analogues (NA) in Europe. METHODS: This study analyzed 828 chronically HBV-infected European patients exposed to ≥ 1 NA, with detectable HBV-DNA and with an available HBsAg-sequence. The immune-associated escape mutations and the NA-induced immune-escape mutations sI195M, sI196S, and sE164D (resulting from drug-resistance mutation rtM204 V, rtM204I, and rtV173L) were retrieved from literature and examined. Mutations were defined as an aminoacid substitution with respect to a genotype A or D reference sequence. RESULTS: At least one immune-associated escape mutation was detected in 22.1% of patients with rising temporal-trend. By multivariable-analysis, genotype-D correlated with higher selection of ≥ 1 immune-associated escape mutation (OR[95%CI]:2.20[1.32-3.67], P = 0.002). In genotype-D, the presence of ≥ 1 immune-associated escape mutations was significantly higher in drug-exposed patients with drug-resistant strains than with wild-type virus (29.5% vs 20.3% P = 0.012). Result confirmed by analysing drug-naïve patients (29.5% vs 21.2%, P = 0.032). Strong correlation was observed between sP120T and rtM204I/V (P < 0.001), and their co-presence determined an increased HBV-DNA. At least one NA-induced immune-escape mutation occurred in 28.6% of patients, and their selection correlated with genotype-A (OR[95%CI]:2.03[1.32-3.10],P = 0.001). Finally, stop-codons are present in 8.4% of patients also at HBsAg-positions 172 and 182, described to enhance viral oncogenic-properties. CONCLUSIONS: Immune-escape mutations and stop-codons develop in a large fraction of NA-exposed patients from Europe. This may represent a potential threat for horizontal and vertical HBV transmission also to vaccinated persons, and fuel drug-resistance emergence.

DMDtoolkit: a tool for visualizing the mutated dystrophin protein and predicting the clinical severity in DMD.

BACKGROUND: Dystrophinopathy is one of the most common human monogenic diseases which results in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD). Mutations in the dystrophin gene are responsible for both DMD and BMD. However, the clinical phenotypes and treatments are quite different in these two muscular dystrophies. Since early diagnosis and treatment results in better clinical outcome in DMD it is essential to establish accurate early diagnosis of DMD to allow efficient management. Previously, the reading-frame rule was used to predict DMD versus BMD. However, there are limitations using this traditional tool. Here, we report a novel molecular method to improve the accuracy of predicting clinical phenotypes in dystrophinopathy. We utilized several additional molecular genetic rules or patterns such as "ambush hypothesis", "hidden stop codons" and "exonic splicing enhancer (ESE)" to predict the expressed clinical phenotypes as DMD versus BMD. RESULTS: A computer software "DMDtoolkit" was developed to visualize the structure and to predict the functional changes of mutated dystrophin protein. It also assists statistical prediction for clinical phenotypes. Using the DMDtoolkit we showed that the accuracy of predicting DMD versus BMD raised about 3% in all types of dystrophin mutations when compared with previous methods. We performed statistical analyses using correlation coefficients, regression coefficients, pedigree graphs, histograms, scatter plots with trend lines, and stem and leaf plots. CONCLUSIONS: We present a novel DMDtoolkit, to improve the accuracy of clinical diagnosis for DMD/BMD. This computer program allows automatic and comprehensive identification of clinical risk and allowing them the benefit of early medication treatments. DMDtoolkit is implemented in Perl and R under the GNU license. This resource is freely available at http://github.com/zhoujp111/DMDtoolkit , and http://www.dmd-registry.com .

The combinatorics of overlapping genes.

Overlapping genes exist in all domains of life and are much more abundant than expected upon their first discovery in the late 1970s. Assuming that the reference gene is read in frame +0, an overlapping gene can be encoded in two reading frames in the sense strand, denoted by +1 and +2, and in three reading frames in the opposite strand, denoted by -0, -1, and -2. This motivated numerous researchers to study the constraints induced by the genetic code on the various overlapping frames, mostly based on information theory. Our focus in this paper is on the constraints induced on two overlapping genes in terms of amino acids, as well as polypeptides. We show that simple linear constraints bind the amino-acid composition of two proteins encoded by overlapping genes. Novel constraints are revealed when polypeptides are considered, and not just single amino acids. For example, in double-coding sequences with an overlapping reading frame -2, each Tyrosine (denoted as Tyr or Y) in the overlapping frame overlaps a Tyrosine in the reference frame +0 (and reciprocally), whereas specific words (e.g. YY) never occur. We thus distinguish between null constraints (YY = 0 in frame -2) and non-null constraints (Y in frame +0 ⇔ Y in frame -2). Our equivalence-based constraints are symmetrical and thus enable the characterization of the joint composition of overlapping proteins. We describe several formal frameworks and a graph algorithm to characterize and compute these constraints. As expected, the degrees of freedom left by these constraints vary drastically among the different overlapping frames. Interestingly, the biological meaning of constraints induced on two overlapping proteins (hydropathy, forbidden di-peptides, expected overlap length …) is also specific to the reading frame. We study the combinatorics of these constraints for overlapping polypeptides of length n, pointing out that, (i) except for frame -2, non-null constraints are deduced from the amino-acid (length = 1) constraints and (ii) null constraints are deduced from the di-peptide (length = 2) constraints. These results yield support for understanding the mechanisms and evolution of overlapping genes, and for developing novel overlapping gene detection methods.

Detection of premature stop codons leading to truncated internalin A among food and clinical strains of Listeria monocytogenes.

Listeria monocytogenes is a food-borne pathogen responsible for outbreaks and sporadic cases of listeriosis, a severe invasive disease. Internalin A (InlA) a protein encoded by inlA has a key role in the mechanism of pathogenesis in L. monocytogenes infection, specifically in the invasion of human intestinal epithelial cells. Studies on inlA have shown that mutations leading to premature stop codons (PMSCs) occur naturally and are associated with impaired virulence of L. monocytogenes strains. Increasing evidence suggests that inlA PMSCs mutations are frequent in strains from foods, but rare among clinical isolates. In this study, 22 L. monocytogenes strains collected in Portugal from the processing environment of a bakery industry (n = 1), different food products (n = 10) and human clinical cases (n = 11) were analysed for mutations in inlA and invasion efficiency in Caco-2 cells. Sequencing revealed previously reported mutations types leading to PMSCs in three food and one clinical strain presenting different molecular serotypes (i.e., IIa, IIb and IIc). The remaining 18 isolates did not show PMSCs in inlA. The four strains with PMSCs in inlA presented lower invasiveness efficiencies in Caco-2 cells (below 8.9%) when compared to the control strain (full-length InlA). In addition, one clinical isolate showed reduced invasion efficiency but no PMSCs in inlA. This isolate showed increased inlA transcript levels to that obtained for the laboratory control strain. Our data support the hypothesis that L. monocytogenes isolated from food have attenuated invasion due to the presence of inlA PMSCs. This information would be critically needed for adequate risk-assessments of the foodborne illness burden associated with L. monocytogenes strains.

Driving through stop signs: predicting stop codon reassignment improves functional annotation of bacteriophages.

The majority of bacteriophage diversity remains uncharacterized, and new intriguing mechanisms of their biology are being continually described. Members of some phage lineages, such as the Crassvirales, repurpose stop codons to encode an amino acid by using alternate genetic codes. Here, we investigated the prevalence of stop codon reassignment in phage genomes and its subsequent impacts on functional annotation. We predicted 76 genomes within INPHARED and 712 vOTUs from the Unified Human Gut Virome Catalogue (UHGV) that repurpose a stop codon to encode an amino acid. We re-annotated these sequences with modified versions of Pharokka and Prokka, called Pharokka-gv and Prokka-gv, to automatically predict stop codon reassignment prior to annotation. Both tools significantly improved the quality of annotations, with Pharokka-gv performing best. For sequences predicted to repurpose TAG to glutamine (translation table 15), Pharokka-gv increased the median gene length (median of per genome median) from 287 to 481 bp for UHGV sequences (67.8% increase) and from 318 to 550 bp for INPHARED sequences (72.9% increase). The re-annotation increased median coding capacity from 66.8% to 90.0% and from 69.0% to 89.8% for UHGV and INPHARED sequences predicted to use translation table 15. Furthermore, the proportion of genes that could be assigned functional annotation increased, including an increase in the number of major capsid proteins that could be identified. We propose that automatic prediction of stop codon reassignment before annotation is beneficial to downstream viral genomic and metagenomic analyses.

Gene-knockout by iSTOP enables rapid reproductive disease modeling and phenotyping in germ cells of the founder generation.

Cytosine base editing achieves C•G-to-T•A substitutions and can convert four codons (CAA/CAG/CGA/TGG) into STOP-codons (induction of STOP-codons, iSTOP) to knock out genes with reduced mosaicism. iSTOP enables direct phenotyping in founders' somatic cells, but it remains unknown whether this works in founders' germ cells so as to rapidly reveal novel genes for fertility. Here, we initially establish that iSTOP in mouse zygotes enables functional characterization of known genes in founders' germ cells: Cfap43-iSTOP male founders manifest expected sperm features resembling human "multiple morphological abnormalities of the flagella" syndrome (i.e., MMAF-like features), while oocytes of Zp3-iSTOP female founders have no zona pellucida. We further illustrate iSTOP's utility for dissecting the functions of unknown genes with Ccdc183, observing MMAF-like features and male infertility in Ccdc183-iSTOP founders, phenotypes concordant with those of Ccdc183-KO offspring. We ultimately establish that CCDC183 is essential for sperm morphogenesis through regulating the assembly of outer dynein arms and participating in the intra-flagellar transport. Our study demonstrates iSTOP as an efficient tool for direct reproductive disease modeling and phenotyping in germ cells of the founder generation, and rapidly reveals the essentiality of Ccdc183 in fertility, thus providing a time-saving approach for validating genetic defects (like nonsense mutations) for human infertility.

Therapeutic Nonsense Suppression Modalities: From Small Molecules to Nucleic Acid-Based Approaches.

Nonsense mutations are genetic mutations that create premature termination codons (PTCs), leading to truncated, defective proteins in diseases such as cystic fibrosis, neurofibromatosis type 1, Dravet syndrome, Hurler syndrome, Beta thalassemia, inherited bone marrow failure syndromes, Duchenne muscular dystrophy, and even cancer. These mutations can also trigger a cellular surveillance mechanism known as nonsense-mediated mRNA decay (NMD) that degrades the PTC-containing mRNA. The activation of NMD can attenuate the consequences of truncated, defective, and potentially toxic proteins in the cell. Since approximately 20% of all single-point mutations are disease-causing nonsense mutations, it is not surprising that this field has received significant attention, resulting in a remarkable advancement in recent years. In fact, since our last review on this topic, new examples of nonsense suppression approaches have been reported, namely new ways of promoting the translational readthrough of PTCs or inhibiting the NMD pathway. With this review, we update the state-of-the-art technologies in nonsense suppression, focusing on novel modalities with therapeutic potential, such as small molecules (readthrough agents, NMD inhibitors, and molecular glue degraders); antisense oligonucleotides; tRNA suppressors; ADAR-mediated RNA editing; targeted pseudouridylation; and gene/base editing. While these various modalities have significantly advanced in their development stage since our last review, each has advantages (e.g., ease of delivery and specificity) and disadvantages (manufacturing complexity and off-target effect potential), which we discuss here.

Designing Guide-RNA for Generating Premature Stop Codons for Gene Knockout Using CRISPR-BETS.

Cytosine base editors (CBEs) accurately modify target sites by mediating a C to T change (or a G to A change on the opposite strand). This allows us to install premature stop codons for gene knockout. However, highly specific sgRNAs (single-guide RNAs) are necessary for the CRISPR-Cas nuclease to work efficiently. In this study, we introduce a method of designing highly specific gRNA to generate premature stop codons and knock out a gene using CRISPR-BETS software.

T-G-A Deficiency Pattern in Protein-Coding Genes and Its Potential Reason.

If a stop codon appears within one gene, then its translation will be terminated earlier than expected. False folding of premature protein will be adverse to the host; hence, all functional genes would tend to avoid the intragenic stop codons. Therefore, we hypothesize that there will be less frequency of nucleotides corresponding to stop codons at each codon position of genes. Here, we validate this inference by investigating the nucleotide frequency at a large scale and results from 19,911 prokaryote genomes revealed that nucleotides coinciding with stop codons indeed have the lowest frequency in most genomes. Interestingly, genes with three types of stop codons all tend to follow a T-G-A deficiency pattern, suggesting that the property of avoiding intragenic termination pressure is the same and the major stop codon TGA plays a dominant role in this effect. Finally, a positive correlation between the TGA deficiency extent and the base length was observed in start-experimentally verified genes of Escherichia coli (E. coli). This strengthens the proof of our hypothesis. The T-G-A deficiency pattern observed would help to understand the evolution of codon usage tactics in extant organisms.

Identification and characterization of novel elastin gene mutations in eleven families with supravalvular aortic stenosis.

Background: Supravalvular aortic stenosis (SVAS) is a rare congenital heart disease affecting approximately 1 in 25,000 live births. In some patients it is accompanied by pulmonary artery stenosis, particularly of pulmonary artery branches. Chronic stenosis can lead to cardiac hypertrophy and even circulatory failure. Familial autosomal dominant SVAS is frequently associated with elastin (ELN) gene mutations, whereas Williams-Beuren syndrome is a complex developmental disorder caused by heterozygous microdeletions of 26-28 genes at 7q11.23, including ELN. Methods: Whole-exome sequencing was performed in 42 individuals from 11 Chinese families with SVAS to identify the pathogenic gene mutations involved. Aortic tissue was obtained for histological analyses, and quantitative reverse-transcription-PCR and western blotting were used to verify the expression of elastin molecules. Results: Five point mutations and six frameshift mutations in the ELN gene were detected in the peripheral blood of all investigated families. Nine were nonsense mutations that result in premature stop codons, and the other two were missense mutations. All variants were heterozygous. Nine of the variants were novel, and have not been included in databases or previously reported. One mutation occurred in individuals from two different families. Reduced elastin protein expression was evident in patients' aortic tissue. Conclusions: The novel mutations of ELN were found to be pathogenic, which confirmed by reduced elastin expression and leads to SVAS. Thus, detailed cardiac testing and genetic counseling are warranted for patients and asymptomatic individuals with these mutations.