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Frontiers in Immunology 2019Using computer algorithms and commercial DNA synthesis, one or more ORFs of a microbial pathogen such as a virus can be recoded and deoptimized by several strategies... (Review)
Review
Using computer algorithms and commercial DNA synthesis, one or more ORFs of a microbial pathogen such as a virus can be recoded and deoptimized by several strategies that may involve the introduction of up to thousands of nucleotide (nt) changes without affecting amino acid (aa) coding. The synonymous recoding strategies that have been applied to RNA viruses include: deoptimization of codon or codon-pair usage, which may reduce protein expression among other effects; increased content of immunomodulatory CpG and UpA RNA, which increase immune responses and thereby restrict viral replication; and substitution of serine and leucine codons with synonymous codons for which single-nt substitutions can yield nonsense codons, thus limiting evolutionary potential. This can reduce pathogen fitness and create potential live-attenuated vaccines that may have improved properties. The combined approach of genome recoding, synthetic biology, and reverse genetics offers several advantages for the generation of attenuated RNA viruses. First, synonymous recoding involves many mutations, which should reduce the rate and magnitude of de-attenuation. Second, increasing the amount of recoding can provide increased attenuation. Third, because there are no changes at the aa level, all of the relevant epitopes should be expressed. Fourth, attenuation frequently does not compromise immunogenicity, suggesting that the recoded viruses have increased immunogenicity per infectious particle. Synonymous deoptimization approaches have been applied to two important human viral pathogens, namely respiratory syncytial virus (RSV) and influenza A virus (IAV). This manuscript will briefly review the use of these different methods of synonymous recoding to generate attenuated RSV and IAV strains. It also will review the characterization of these vaccine candidates and in animal models, and describe several surprising findings with respect to phenotypic and genetic instability of some of these candidates.
Topics: Amino Acid Substitution; Animals; Base Composition; Codon; Genome, Viral; Humans; Respiratory Tract Infections; Silent Mutation; Vaccinology; Viral Proteins; Viral Vaccines; Virus Replication; Viruses
PubMed: 31231383
DOI: 10.3389/fimmu.2019.01250 -
Journal of Personalized Medicine Feb 2024Hypodontia, i.e., missing one or more teeth, is a relatively common human disease; however, oligodontia, i.e., missing six or more teeth, excluding the third molars, is...
Hypodontia, i.e., missing one or more teeth, is a relatively common human disease; however, oligodontia, i.e., missing six or more teeth, excluding the third molars, is a rare congenital disorder. Many genes have been shown to cause oligodontia in non-syndromic or syndromic conditions. In this study, we identified two novel mutations in two non-syndromic oligodontia families. A mutational analysis identified a silent mutation (NM_006194.4: c.771G>A, p.(Gln257=)) in family 1 and a frameshift mutation caused by a single nucleotide duplication (c.637dup, p.(Asp213Glyfs*104)) in family 2. A minigene splicing assay revealed that the silent mutation resulted in aberrant pre-mRNA splicing instead of normal splicing. The altered splicing products are ones with an exon 4 deletion or using a cryptic 5' splicing site in exon 4. Mutational effects were further investigated using protein expression, luciferase activity assay and immunolocalization. We believe this study will not only expand the mutational spectrum of mutations in oligodontia but also strengthen the diagnostic power related to the identified silent mutation.
PubMed: 38392624
DOI: 10.3390/jpm14020191 -
Journal of Cellular and Molecular... Sep 2023Hairy cell leukaemia (HCL) diagnosis is based on the morphologic detection of circulating abnormal hairy cells in the peripheral blood and/or bone marrow, an HCL...
Hairy cell leukaemia (HCL) diagnosis is based on the morphologic detection of circulating abnormal hairy cells in the peripheral blood and/or bone marrow, an HCL immunological score of 3 or 4 based on the expression of the CD11c, CD25, CD103 and CD123 and also the presence of a BRAF V600E activating mutation in the B-raf proto-oncogene (BRAF gene) (7q34). When using new generation sequencing of 21 targeted genes in 124 HCL patients, we identified a cohort of 6/124 (2%) patients with unusual BRAF mutations: two patients presented non-V600 mutations (BRAF F595L, BRAF W604L respectively) and four other patients silent BRAF mutations. When using droplet digital PCR (ddPCR) three of the four patients with concomitant BRAF V600E and silent mutation were negative. The respective role of these mutations in the occurrence of HCL or its progression remains to be clarified, but BRAF sequencing is necessary in case of negative BRAF V600E by ddPCR.
Topics: Humans; Leukemia, Hairy Cell; Proto-Oncogene Proteins B-raf; Mutation; Bone Marrow; Exons
PubMed: 37530550
DOI: 10.1111/jcmm.17890 -
British Journal of Cancer Aug 2023Large cell lung carcinoma (LCLC) is an exceptionally aggressive disease with a poor prognosis. At present, little is known about the molecular pathology of LCLC.
BACKGROUND
Large cell lung carcinoma (LCLC) is an exceptionally aggressive disease with a poor prognosis. At present, little is known about the molecular pathology of LCLC.
METHODS
Ultra-deep sequencing of cancer-related genes and exome sequencing were used to detect the LCLC mutational in 118 tumor-normal pairs. The cell function test was employed to confirm the potential carcinogenic mutation of PI3K pathway.
RESULTS
The mutation pattern is determined by the predominance of A > C mutations. Genes with a significant non-silent mutation frequency (FDR) < 0.05) include TP53 (47.5%), EGFR (13.6%) and PTEN (12.1%). Moreover, PI3K signaling (including EGFR, FGRG4, ITGA1, ITGA5, and ITGA2B) is the most mutated pathway, influencing 61.9% (73/118) of the LCLC samples. The cell function test confirmed that the potential carcinogenic mutation of PI3K pathway had a more malignant cell function phenotype. Multivariate analysis further revealed that patients with the PI3K signaling pathway mutations have a poor prognosis (P = 0.007).
CONCLUSIONS
These results initially identified frequent mutation of PI3K signaling pathways in LCLC and indicate potential targets for the treatment of this fatal type of LCLC.
Topics: Humans; Phosphatidylinositol 3-Kinases; Exome; Carcinoma, Non-Small-Cell Lung; Mutation; Lung Neoplasms; Carcinoma, Large Cell; ErbB Receptors; Lung
PubMed: 37179440
DOI: 10.1038/s41416-023-02301-2 -
Kidney360 Mar 2022Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many...
BACKGROUND
Alport syndrome is an inherited disorder characterized by progressive renal disease, variable sensorineural hearing loss, and ocular abnormalities. Although many pathogenic variants in and have been identified in patients with autosomal Alport syndrome, synonymous mutations in these genes have rarely been identified.
METHODS
We conducted splicing analysis using Human Splicing Finder (HSF) and Alamut to predict splicing domain strength and disruption of the sites. Furthermore, we performed splicing assays using minigene constructs and mRNA analysis of patient samples to determine the pathogenicity of four synonymous variants detected in four patients with suspected autosomal dominant Alport syndrome ( [c.693G>A (p.Val231=)] and [c.1353C>T (p.Gly451=), c.735G>A (p.Pro245=), and c.870G>A (p.Lys290=)]).
RESULTS
Both and splicing assays showed exon skipping in two out of the four synonymous variants identified (c.735G>A and c.870G>A in ). Prediction analysis of wild-type and mutated sequences using HSF and Alamut suggested these two variants may lead to the loss of binding sites for several splicing factors, , in acceptor sites and exonic splicing enhancers. The other two variants did not induce aberrant splicing.
CONCLUSIONS
This study highlights the pitfalls of classifying the functional consequences of variants by a simple approach. Certain synonymous variants, although they do not alter the amino acid sequence of the encoded protein, can dramatically affect pre-mRNA splicing, as shown in two of our patients. Our findings indicate that transcript analysis should be carried out to evaluate synonymous variants detected in patients with autosomal dominant Alport syndrome.
Topics: Autoantigens; Collagen Type IV; Exons; Humans; Nephritis, Hereditary; Silent Mutation
PubMed: 35582193
DOI: 10.34067/KID.0005252021 -
Biomolecules Jan 2024Synonymous mutations result from the degeneracy of the genetic code. Most amino acids are encoded by two or more codons, and mutations that change a codon to another... (Review)
Review
Synonymous mutations result from the degeneracy of the genetic code. Most amino acids are encoded by two or more codons, and mutations that change a codon to another synonymous codon do not change the amino acid in the gene product. Historically, such mutations have been considered silent because they were assumed to have no to very little impact. However, research in the last few decades has produced several examples where synonymous mutations play important roles. These include optimizing expression by enhancing translation initiation and accelerating or decelerating translation elongation via codon usage and mRNA secondary structures, stabilizing mRNA molecules and preventing their breakdown before translation, and faulty protein folding or increased degradation due to enhanced ubiquitination and suboptimal secretion of proteins into the appropriate cell compartments. Some consequences of synonymous mutations, such as mRNA stability, can lead to different outcomes in prokaryotes and eukaryotes. Despite these examples, the significance of synonymous mutations in evolution and in causing disease in comparison to nonsynonymous mutations that do change amino acid residues in proteins remains controversial. Whether the molecular mechanisms described by which synonymous mutations affect organisms can be generalized remains poorly understood and warrants future research in this area.
Topics: Silent Mutation; Codon; Protein Biosynthesis; RNA, Messenger; Proteins; Amino Acids; Evolution, Molecular
PubMed: 38275761
DOI: 10.3390/biom14010132 -
Scientific Reports Jun 2022Colorectal cancer (CRC) is a heterogeneous disease with evidence of distinct tumor types that develop through different somatically altered pathways. To better...
Colorectal cancer (CRC) is a heterogeneous disease with evidence of distinct tumor types that develop through different somatically altered pathways. To better understand the impact of the host genome on somatically mutated genes and pathways, we assessed associations of germline variations with somatic events via two complementary approaches. We first analyzed the association between individual germline genetic variants and the presence of non-silent somatic mutations in genes in 1375 CRC cases with genome-wide SNPs data and a tumor sequencing panel targeting 205 genes. In the second analysis, we tested if germline variants located within previously identified regions of somatic allelic imbalance were associated with overall CRC risk using summary statistics from a recent large scale GWAS (n≃125 k CRC cases and controls). The first analysis revealed that a variant (rs78963230) located within a CNA region associated with TLR3 was also associated with a non-silent mutation within gene FBXW7. In the secondary analysis, the variant rs2302274 located in CDX1/PDGFRB frequently gained/lost in colorectal tumors was associated with overall CRC risk (OR = 0.96, p = 7.50e-7). In summary, we demonstrate that an integrative analysis of somatic and germline variation can lead to new insights about CRC.
Topics: Allelic Imbalance; Colorectal Neoplasms; Genetic Predisposition to Disease; Germ Cells; Germ-Line Mutation; Humans; Polymorphism, Single Nucleotide
PubMed: 35715570
DOI: 10.1038/s41598-022-14408-2 -
Genome Biology and Evolution Oct 2021Owing to a lag between a deleterious mutation's appearance and its selective removal, gold-standard methods for mutation rate estimation assume no meaningful loss of...
Owing to a lag between a deleterious mutation's appearance and its selective removal, gold-standard methods for mutation rate estimation assume no meaningful loss of mutations between parents and offspring. Indeed, from analysis of closely related lineages, in SARS-CoV-2, the Ka/Ks ratio was previously estimated as 1.008, suggesting no within-host selection. By contrast, we find a higher number of observed SNPs at 4-fold degenerate sites than elsewhere and, allowing for the virus's complex mutational and compositional biases, estimate that the mutation rate is at least 49-67% higher than would be estimated based on the rate of appearance of variants in sampled genomes. Given the high Ka/Ks one might assume that the majority of such intrahost selection is the purging of nonsense mutations. However, we estimate that selection against nonsense mutations accounts for only ∼10% of all the "missing" mutations. Instead, classical protein-level selective filters (against chemically disparate amino acids and those predicted to disrupt protein functionality) account for many missing mutations. It is less obvious why for an intracellular parasite, amino acid cost parameters, notably amino acid decay rate, is also significant. Perhaps most surprisingly, we also find evidence for real-time selection against synonymous mutations that move codon usage away from that of humans. We conclude that there is common intrahost selection on SARS-CoV-2 that acts on nonsense, missense, and possibly synonymous mutations. This has implications for methods of mutation rate estimation, for determining times to common ancestry and the potential for intrahost evolution including vaccine escape.
Topics: COVID-19; Codon Usage; Codon, Nonsense; Evolution, Molecular; Humans; Models, Genetic; Mutation; Mutation Rate; Mutation, Missense; Polymorphism, Single Nucleotide; SARS-CoV-2; Selection, Genetic; Silent Mutation
PubMed: 34427640
DOI: 10.1093/gbe/evab196 -
Journal For Immunotherapy of Cancer Oct 2019The role of tumor-infiltrating B-cells (TIBs) and intratumorally-produced antibodies in cancer-immunity interactions essentially remains terra incognita. In particular,...
BACKGROUND
The role of tumor-infiltrating B-cells (TIBs) and intratumorally-produced antibodies in cancer-immunity interactions essentially remains terra incognita. In particular, it remains unexplored how driver mutations could be associated with distinct TIBs signatures and their role in tumor microenvironment.
METHODS
Here we analyzed associations of immunoglobulin isotypes and clonality with survival in TCGA RNA-Seq data for lung adenocarcinoma (LUAD), stratifying patients into 12 driver mutation and phenotypic tumor subgroups.
RESULTS
We revealed several unexpected associations between TIBs behavior and prognosis. Abundance and high proportion of IgG1 isotype, and low proportion of IgA among all intratumorally produced immunoglobulins were specifically associated with improved overall survival for KRAS but not KRAS LUAD, revealing the first link between a driver mutation and B-cell response. We found specific IgG1 signature associated with long survival, which suggests that particular specificities of IgG1+ TIBs could be beneficial in KRAS LUAD. In contrast to our previous observations for melanoma, highly clonal IgG1 production by plasma cells had no meaningful effect on prognosis, suggesting that IgG1+ TIBs may exert a beneficial effect in KRAS cases in an alternative way, such as efficient presentation of cognate antigens or direct B cell attack on tumor cells. Notably, a high proportion of the IgG1 isotype is positively correlated with the non-silent mutation burden both in the general LUAD cohort and in most patient subgroups, supporting a role for IgG1 TIBs in antigen presentation. Complementing the recent finding that the presence of stromal IgG4-producing cells is associated with a favorable prognosis for patients with stage I squamous cell carcinoma, we show that the abundance of IgG4-producing TIBs likewise has a strong positive effect on overall survival in STK11 and proximal proliferative subgroups of LUAD patients. We hypothesize that the positive role of IgG4 antibodies in some of the lung cancer subtypes could be associated with reported inability of IgG4 isotype to form immune complexes, thus preventing immunosuppression via activation of the myeloid-derived suppressor cell (MDSC) phenotype.
CONCLUSIONS
We discover prominent and distinct associations between TIBs antibody isotypes and survival in lung adenocarcinoma carrying specific driver mutations. These findings indicate that particular types of tumor-immunity relations could be beneficial in particular driver mutation context, which should be taken into account in developing strategies of cancer immunotherapy and combination therapies. Specificity of protective B cell populations in specific cancer subgroups could become a clue to efficient targeted immunotherapies for appropriate cohorts of patients.
Topics: Adenocarcinoma of Lung; Antibody Formation; B-Lymphocytes; Biomarkers, Tumor; Databases, Factual; Gene Expression; Humans; Immunoglobulin Isotypes; Kaplan-Meier Estimate; Lung Neoplasms; Mutation; Prognosis; Tumor Microenvironment
PubMed: 31665076
DOI: 10.1186/s40425-019-0747-1 -
Genome Biology and Evolution Sep 2021Protein coding genes can contain specific motifs within their nucleotide sequence that function as a signal for various biological pathways. The presence of such... (Review)
Review
Protein coding genes can contain specific motifs within their nucleotide sequence that function as a signal for various biological pathways. The presence of such sequence motifs within a gene can have beneficial or detrimental effects on the phenotype and fitness of an organism, and this can lead to the enrichment or avoidance of this sequence motif. The degeneracy of the genetic code allows for the existence of alternative synonymous sequences that exclude or include these motifs, while keeping the encoded amino acid sequence intact. This implies that locally, there can be a selective pressure for preferentially using a codon over its synonymous alternative in order to avoid or enrich a specific sequence motif. This selective pressure could-in addition to mutation, drift and selection for translation efficiency and accuracy-contribute to shape the codon usage bias. In this review, we discuss patterns of avoidance of (or enrichment for) the various biological signals contained in specific nucleotide sequence motifs: transcription and translation initiation and termination signals, mRNA maturation signals, and antiviral immune system targets. Experimental data on the phenotypic or fitness effects of synonymous mutations in these sequence motifs confirm that they can be targets of local selection pressures on codon usage. We also formulate the hypothesis that transposable elements could have a similar impact on codon usage through their preferred integration sequences. Overall, selection on codon usage appears to be a combination of a global selection pressure imposed by the translation machinery, and a patchwork of local selection pressures related to biological signals contained in specific sequence motifs.
Topics: Codon; Codon Usage; Genetic Code; Mutation; Selection, Genetic; Silent Mutation
PubMed: 33944930
DOI: 10.1093/gbe/evab097