Silent Mutations Research Articles

A Comparison of Bovine Torovirus Strains Based on Partial Membrane Glycoprotein (M) Gene Sequences

Bovine torovirus (BoTV), which was recently been separated from bovine coronaviruses, is one of the important causative agents of diarrhea in newborn calves. Although the epidemiological data are limited throughout the world, it has been reported in some countries of the world since the early 80’s. In this study, stool samples (n: 150) were taken from 0-30 days old diarrhoeic calves from Elazig, Sivas, and Malatya provinces and were screened by nested RT-PCR method. In 6% (9/150) of the studied samples, 409 bp partial membrane glycoprotein (M) gene was successfully amplified. Some of the positive samples were further sequenced and the 383 nt length data of nine samples were subjected to bioinformatic analysis. The multiple sequence comparison and phylogenetic analyses together revealed that these novel strains presented close identity to previously reported strains from Turkey. The nucleotide identity of the strains was found between 97.13% and 100%. Furthermore, valine-isoleucine substitution (V→I) at 114th position was detected in Turkish strains only (MN717266; MF687255-60), whereas the same substitution at the 144th position was shared between the Turkish (KF188708-11 and KF188714; MF687255-60; MG957145 and MN717266-67) and some of the Chinese-originated isolates. Moreover, four silent mutations were detected in the novel isolates subjected in this study. As a result, we demonstrated the contribution of BToV in the pathogenesis of calf diarrhea and presented new data concerning on the molecular characteristics and the epidemiology of the bovine toroviruses in Turkey.

Two Individuals with Rare Blocked Antigen Phenomenon and Coinciding Warm Autoantibody Mimicking Alloanti-Jk3 Resolved with JK Analysis

Abstract Introduction/Objective Kidd antigens can bind complement (C3) as well as Kidd specific warm autoantibodies (WAAb). An 838G>A single nucleotide variant (SNV) defines JK*01 and JK*02 which codes the antithetical Jka and Jk b, respectively. Both alleles translate the high prevalence (>99%) Jk3 (JK3). The 130G>A is associated with weak Jka and weak Jkb expression. In vivo binding of non-agglutinating globulins can cause false-negative phenotypes by means of the blocked antigen phenomenon (BAP). Methods/Case Report Transfusions were requested for a 74-year-old Caucasian (CA) female with Evan’s Syndrome, and an 85-year-old African American (AA) female with metastatic uterine cancer. Both had a history of nonspecific WAAb. Direct antiglobulin testing (DAT) detected moderate in vivo sensitization of IgG and C3. They phenotyped Jk(a- b-) with untreated and EDTA glycine-acid (EGA) treated IgG DAT-negative cells. Their serum contained anti-Jk3 reactivity, while a panreactive WAAb in the eluate reacted with Jk3- donor and EGA treated DAT-negative autologous cells. Weak anti-Jka and anti-Jkb reactivity remained in the alloadsorbed serum of the antithetical adsorbing cells. Genetic testing of the CA revealed JK*01W.01(130A)/02 alleles, while cDNA confirmed the alleles would be transcribed into mRNA. Sequencing of the AA detected 130G/A, and 838G/A as well as other silent mutations predicting either a Jk(a+wb+) or Jk(a+b+w) phenotype. The CA received one compatible JK:-3 transfusion, and both individuals benefited from multiple least incompatible transfusions of Jk a+ and/or Jk b+ donors with expected hemoglobin increases (1 g/dL per transfusion). The CA serologically phenotyped Jk(a-b+) 132 days later following prolonged immunosuppressive therapy while a normocytic normochromic anemia and the WAAb persisted. No follow up evaluations of the AA are available. Results (if a Case Study enter NA) NA Conclusion Unexpected BAP can confound immunohematology testing and lead WAAbs mimicking alloanti-Jk3 to be mischaracterized as allogeneic. By predicting phenotypes, genetic analysis can aid serological techniques in antibody characterization and help circumvent complications searching for rare JK:-3 donors.

Construction of an Influenza D Virus with an Eight-Segmented Genome.

Influenza D virus (IDV) may cause the bovine respiratory disease complex, which is the most common and costly disease affecting the cattle industry. Previously, we revealed that eight segments could be actively packaged in its single virion, suggesting that IDV with the seven-segmented genome shows an agnostic genome packaging mechanism. Herein, we engineered an eight-segmented recombinant IDV in which the NS1 or NS2 genes were separated from NS segment into independent segments (NS1 or NS2 segments, respectively), leading to monocistronic translation of each NS protein. We constructed two plasmids: one for the viral RNA (vRNA)-synthesis of the NS1 segment with a silent mutation at the splicing acceptor site, which controls NS2 transcription in the NS segment; and another for the RNA synthesis of the NS2 segment, with deletion of the intron in the NS segment. These plasmids and six other vRNA-synthesis plasmids were used to fabricate an infectious eight-segmented IDV via reverse genetics. This system enables analysis of the functions of NS1 or NS2. We tested the requirement of the N-terminal overlapping region (NOR) in these proteins for viral infectivity. We rescued a virus with NOR-deleted NS2 protein, which displayed a growth rate equivalent to that of the eight-segmented virus with intact NS2. Thus, the NOR may not influence viral growth. In contrast, a virus with NOR-deleted NS1 protein could not be rescued. These results indicate that the eight-segmented rescue system of IDV may provide an alternative method to analyze viral proteins at the molecular level.

Determination of mucopolysaccharidosis IIID in some goat breeds

Mucopolysaccharidosis IIID (MPS IIID) or Sanfilippo D syndrome is an inherited lysosomal storage disease and caused by a deficiency of the N-acetylglucosamine-6-sulfatase activity, which involves in the catabolism of heparan sulfate. In addition to humans, canine, feline and murine, goats have been reported as possessing this lysosomal storage disease. The molecular defect in the MPS IIID goat has been previously identified. It has been observed that a molecular base for this defect is a nonsense mutation at nucleotide 322 (C→T) results in the change of the arginine codon to a stop codon. This mutation is not only to be related to stop enzyme function but also introduces a recognition site for AluI that will enable carrier detection. The goal of this study is to find the presence of MPS IIID genetic disorder in some goat breeds. A total of 80 goat blood samples selected randomly from 4 different countries including Kyrgyzstan, Iraq, Northern Cyprus and Bosnia-Herzegovina were used as a sample. Polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) and DNA sequence data were utilized to identify this disorder in the goat populations. The result of this study demonstrates nonsense mutation which causes MPS IIID genetic disorder was not found in the goat breeds studied, while a silent mutation was found at nucleotide 354 (T→C) when compared with reference sequence.

A mutational hotspot that determines highly repeatable evolution can be built and broken by silent genetic changes

Mutational hotspots can determine evolutionary outcomes and make evolution repeatable. Hotspots are products of multiple evolutionary forces including mutation rate heterogeneity, but this variable is often hard to identify. In this work, we reveal that a near-deterministic genetic hotspot can be built and broken by a handful of silent mutations. We observe this when studying homologous immotile variants of the bacteria Pseudomonas fluorescens, AR2 and Pf0-2x. AR2 resurrects motility through highly repeatable de novo mutation of the same nucleotide in >95% lines in minimal media (ntrB A289C). Pf0-2x, however, evolves via a number of mutations meaning the two strains diverge significantly during adaptation. We determine that this evolutionary disparity is owed to just 6 synonymous variations within the ntrB locus, which we demonstrate by swapping the sites and observing that we are able to both break (>95% to 0%) and build (0% to 80%) a deterministic mutational hotspot. Our work reveals a key role for silent genetic variation in determining adaptive outcomes.

Bongard and Smirnov on the tetrachromacy of extra-foveal vision

In Moscow in the 1950′s, the physicist M. M. Bongard developed the use of silent substitution to establish the number of dimensions of human or animal colour vision and to derive colour-matching functions either for whole organisms or for individual neuronal channels. In 1956, he and his colleague M. S. Smirnov reported that extra-foveal human vision was tetrachromatic when tested by the silent-substitution method that they called ‘replacement colorimetry’. In the steady state, trichromatic matches were possible in extra-foveal regions, but transients were visible when one such match was replaced by another. If, however, a match was made with four primaries, then a silent substitution was possible; and such matches – unlike trichromatic ones – were stable with light level and with changes in the state of chromatic adaptation. Bongard and Smirnov believed that the fourth receptor had the spectral sensitivity of the rods, but of course they were working long before the discovery of intrinsically photosensitive retinal ganglion cells. On the fiftieth anniversary of Bongard’s grievous death, we provide a translation of Bongard and Smirnov’s paper on the tetrachromacy of extra-foveal vision. In a commentary, we give the background to their work and provide further details of their apparatus and procedure. We briefly discuss related research and the reception in the West of Bongard and Smirnov’s claims. We suggest that an analogy can be made between the tetrachromacy of the parafovea and the ‘weak tetrachromacy’ of heterozygotes for anomalous colour vision, whose trichromatic matches are not stable with chromatic adaptation.

Genetic characterization of genotype VII.1.1 Newcastle Disease viruses from commercial and backyard broiler chickens in Egypt

The small-scale and backyard-raised poultry are extensively growing in Egypt. However, low biosecurity and/or vaccination practices are adopted. The current study was conducted to investigate and molecularly characterize Newcastle Disease (ND) strains circulating in small-scale and backyard poultry sectors in Giza governorate in Egypt from July 2018 to April 2020. Twelve broiler flocks (6 commercial and 6 backyard flocks) suffering from respiratory and/or nervous signs were included. Virus isolation and molecular characterization were conducted. The isolated viruses maintained the multi-basic motif 112RRQKR|F117 in the fusion protein cleavage site, indicating their virulent nature. Phylogenetically, the viruses are closely related to genotype VII.1.1. ND viruses and are genetically different from NDV genotype II vaccine strains. Deduced amino acid sequences of the fusion (F) protein showed an amino acid change of S278P in the heptad repeat (HRb) in the two NDV isolates from commercial farms. Other amino acid substitutions were observed, including the change at positions 442 (A/T) in the two commercial flocks isolates and position 317 (A/S) in the backyard ND isolate. Few silent nucleotide mutations were also observed in the F protein of the NDV isolate from the backyard flock. In conclusion, the current study reports the genetic identification of virulent NDV viruses from commercial or backyard flocks related to genotype VII.1.1. ND circulating in Egypt. The amino acid substitutions and nucleotide changes warrant the need for continuous surveillance of NDV in these growing poultry sectors considering their importance as a spot for extensive NDV circulation and as the main source for live bird markets in Egypt.

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

SummaryWhile prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types.

Rescuing eGFP-Tagged Canine Distemper Virus for 40 Serial Passages Separately in Ribavirin- and Non-Treated Cells: Comparative Analysis of Viral Mutation Profiles.

Due to lacking a proofreading mechanism in their RNA-dependent RNA polymerases (RdRp), RNA viruses generally possess high mutation frequencies, making them evolve rapidly to form viral quasispecies during serial passages in cells, especially treated with mutagens, like ribavirin. Canine distemper virus (CDV) belongs to the genus Morbillivirus. Its L protein functions as an RdRp during viral replication. In this study, a recombinant enhanced green fluorescence protein-tagged CDV (rCDV-eGFP) was rescued from its cDNA clone, followed by viral identification and characterization at passage-7 (P7). This recombinant was independently subjected to extra 40 serial passages (P8 to 47) in ribavirin- and non-treated cells. Two viral progenies, undergoing passages in ribavirin- and non-treated VDS cells, were named rCDV-eGFP-R and -N, respectively. Both progenies were simultaneously subjected to next-generation sequencing (NGS) at P47 for comparing their quasispecies diversities with each other. The rCDV-eGFP-R and -N showed 62 and 23 single-nucleotide mutations (SNMs) in individual antigenomes, respectively, suggesting that the ribavirin conferred a mutagenic effect on the rCDV-eGFP-R. The spectrum of 62 SNMs contained 26 missense and 36 silent mutations, and that of 23 SNMs was composed of 17 missense and 6 silent mutations. Neither the rCDV-eGFP-R nor -N exhibited nonsense mutation in individual antigenomes. We speculate that the rCDV-eGFP-R may contain at least one P47 sub-progeny characterized by high-fidelity replication in cells. If such a sub-progeny can be purified from the mutant swarm, its L protein would elucidate a molecular mechanism of CDV high-fidelity replication.

Role of NSD1 as potential therapeutic target in tumor

Nuclear receptor binding SET Domain Protein 1 (NSD1) is a bifunctional transcriptional regulatory protein that encodes histone methyltransferase. Mono- and di-methylation of H3K36 by NSD1 is mainly primarily involved in the regulation of gene expression, DNA repair, alternative splicing, and other important biological processes. Many types of cancers, including acute myelogenous leukemia (AML), liver cancer, lung cancer, endometrial carcinoma, colorectal cancer, and pancreatic cancer, are associated with NSD1 fusion, missense mutation, nonsense mutation, silent mutation, deletion, and insertion of frameshift, and deletion in a frame. Therefore, targeting NSD1 may be a potential strategy for tumor therapy. An in-depth study of the structure and biological activities of NSD1 sets the groundwork for improving tumor therapy and creating NSD1 inhibitors. This article emphasizes the role of NSD1 in tumorigenesis and the development of NSD1 targeted small-molecule inhibitors.

Genomic variation in captive deer mouse (Peromyscus maniculatus) populations

BackgroundDeer mice (genus Peromyscus) are the most common rodents in North America. Despite the availability of reference genomes for some species, a comprehensive database of polymorphisms, especially in those maintained as living stocks and distributed to academic investigators, is missing. In the present study we surveyed two populations of P. maniculatus that are maintained at the Peromyscus Genetic Stock Center (PGSC) for polymorphisms across their 2.5 × 109 bp genome.ResultsHigh density of variation was identified, corresponding to one SNP every 55 bp for the high altitude stock (SM2) or 207 bp for the low altitude stock (BW) using snpEff (v4.3). Indels were detected every 1157 bp for BW or 311 bp for SM2. The average Watterson estimator for the BW and SM2 populations is 248813.70388 and 869071.7671 respectively. Some differences in the distribution of missense, nonsense and silent mutations were identified between the stocks, as well as polymorphisms in genes associated with inflammation (NFATC2), hypoxia (HIF1a) and cholesterol metabolism (INSIG1) and may possess value in modeling pathology.ConclusionsThis genomic resource, in combination with the availability of P. maniculatus from the PGSC, is expected to promote genetic and genomic studies with this animal model.

Reduced pathogenicity of velogenic NDV strain AF22420-I via site-directed mutagenesis of V gene

Newcastle disease virus (NDV), an avian paramyxovirus, has the potential to be used as an anti-cancer therapeutic vaccine due to its oncolytic and immunostimulatory activities. The virus can be categorised into three pathotypes: lentogenic, mesogenic, and velogenic; of the three pathotypes, the lentogenic strains such as the La Sota are the preferred pathotype for vaccine development due to their low virulence to birds. On the other hand, the translation of the virus to clinic of the velogenic strain AF2240-I is hindered by its virulence towards birds although it exhibits strong oncolysis with significant outcomes both in vitro and in vivo. This study aims to reduce the pathogenicity of AF2240-I yet retaining the anti-cancer properties of the virus. To achieve this, the V protein that acts as an interferon antagonist was chosen to be mutated. It is a non-structural protein that does not interfere with the binding and infection of the virus; hence, mutation of this virulence factor was deducted to be able to reduce harm to the avian species but retain its anti-cancer properties as much as possible. The V protein, which was produced from the insertion of an additional G into a conserved editing site of the P gene, was mutated by substituting the G nucleotide at position 411 from the start of P gene to a T nucleotide. This mutation will produce a premature stop codon from the V mRNA, resulting in a truncated V protein; but only causes a silent mutation in the P protein. The recombinant virus was recovered by the use of BHK cells stably expressing the phage T7 RNA polymerase. The pathogenicity of the mutated virus was determined in 9- to 11-day-old embryonated SPF chicken eggs. The mean death time (MDT) was determined to be 73.6 hours at the minimal lethal dose of 10-7, resembling to that of a mesogenic strain. The virulence of the mutated virus has been successfully reduced where it could be potentially used as the vector for the development of recombinant oncolytic virus for cancer treatment.

Rates and patterns of molecular evolution in bryophyte genomes, with focus on complex thalloid liverworts, Marchantiopsida

Plants commonly referred to as “bryophytes” belong to three major lineages of non-vascular plants: the liverworts, the hornworts and the mosses. They are unique among land plants in having a dominant haploid generation and a short-lived diploid sporophytic generation. The dynamics of selection acting on a haploid genome differs from those acting on a diploid genome: new mutations are directly exposed to selection. The general aim of this paper is to investigate the diversification rateof bryophytes - measured as silent site substitution rate representing neutral evolution (mutation rate) and the nonsynonymous to synonymous substitution rate ratio (dN/dS) representing selective evolution - and compare it with earlier studies on vascular plants. Results show that the silent site substitution rate is lower for liverworts as compared to angiosperms, but not as low as for gymnosperms. The selection pressure, measured as dN/dS, isnot remarkably lower for bryophytes as compared to other diploid dominant plants as would be expected by the masking hypothesis, indicating that other factors are more important than ploidy.

Maximum possible contrast level for silent substitution: a theoretical model applied to melanopsin stimulation.

For any given set of light sources stimulating the photoreceptors of the retina, the theoretical levels of illumination producing the smallest and the largest expression of one photoreceptor with fixed stimulation for the others are analytically computed. The cases of four, five, and more light sources are studied. We show that, for contrast optimization, only as many light sources as photoreceptors do matter and that, in the case of four light sources, the maximum contrast achievable for melanopsin lies at the intersection of the lines joining the sources in the CIE xy chromaticity diagram. This result is used to obtain the optimal position of four Gaussian primaries of equal bandwidth. In addition, we derive a procedure to construct level maps for melanopsin contrast overlying the diagram. In the second part of the paper, the interpersonal variability of the perceived stimulation is shown to be globally reduced if the bandwidth of the light sources is increased and, under some assumptions, if a light source is added.

A novel HLA-B allele, HLA-B*50:04:02, detected in a Russian hematopoietic stem cell donor.

HLA-B*50:04:02 differs from HLA-B*50:04:01 by two silent mutations.

Molecular detection of vicR, vicK and 16S rRNA genes of Streptococcus mutans Isolated from Dental Caries of Iraqi’s Patient

Background : This study was carried out to detect the distribution of Streptococcus mutans with dentalcaries in Iraqi’s patient samples. Out of 100 patient’s samples only (22) with age (3-17) years old werefound to be effected with Streptococcus mutans. DNA was extracted amplified by Gradient PCR and thensequenced. vicR was showed highly conserved with no mutation while vicK showed that these were silentmutation and four isolates have missense mutation from eight isolate.Material & Method : In this study, 22 from clinical S. mutans strains were isolated from caries- childrento sequence the vicR, vicK and 16s rRNA genes. Genomic DNA was extracted from S. mutans strains andamplified using Gradient PCR. The PCR products were purified and then sequenced.Result : The molecular study include vicR, vicK and 16S rRNA, genes by Gradient PCR. The result ofsequencing appear that no mutation in vicR genes, although in 16S rRNA present transion and tranvesionmutation . Finally the sequencing of vicK gene appear silent mutation and four isolates have missensemutation from eightConclusion the result of molecular study of vicR gene were appear no mutation in all isolates while 16SrRNA although have transition and transvertion mutation, the Sequence of vicK gene that note more ofmutation was silent mutation but some of them was missense mutation.

Estimating the predictive power of silent mutations on cancer classification and prognosis

In recent years it has been shown that silent mutations, in and out of the coding region, can affect gene expression and may be related to tumorigenesis and cancer cell fitness. However, the predictive ability of these mutations for cancer type diagnosis and prognosis has not been evaluated yet. In the current study, based on the analysis of 9,915 cancer genomes and approximately three million mutations, we provide a comprehensive quantitative evaluation of the predictive power of various types of silent and non-silent mutations over cancer classification and prognosis. The results indicate that silent-mutation models outperform the equivalent null models in classifying all examined cancer types and in estimating the probability of survival 10 years after the initial diagnosis. Additionally, combining both non-silent and silent mutations achieved the best classification results for 68% of the cancer types and the best survival estimation results for up to nine years after the diagnosis. Thus, silent mutations hold considerable predictive power over both cancer classification and prognosis, most likely due to their effect on gene expression. It is highly advised that silent mutations are integrated in cancer research in order to unravel the full genomic landscape of cancer and its ramifications on cancer fitness.

No Tumor Suppressor Role for LKB1 in Prostate Cancer.

To elucidate the pathogenesis of prostate diseases, following in silico analysis, the LKB1 gene was selected for further investigation. The LKB1 gene has been associated with poor prognosis and is frequently mutated in different types of cancers. In this study, 50 benign prostatic hyperplasia (BPH) and 57 prostate cancer (PCa) tissues, including matched normal tissue for the patients, were analyzed by qRT-PCR and DNA sequencing for LKB1 expression and the mutation profile, respectively. Expression of LKB1 was increased in 60.7% of the PCa tissues compared with noncancerous tissue samples (p ≤ 0.001). However, LKB1 expression was lower when compared with normal tissues in BPH (p = 0.920). Four coding sequence alterations were detected in BPH. Three silent mutations were located in codons 9, 32, and 275 and a missense mutation was observed in codon 384. Six alterations were identified in the intronic regions of the LKB1 gene in both PCa and BPH. Five mutations were observed in both patient groups. A new alteration in intron 6 was observed in a patient with PCa. The LKB1 gene may be associated with benign transformations rather than the tumors in prostate pathogenesis when its expression and mutation status are considered. However, the mechanism of LKB1 in PCa needs further studies.

Characterization of altered genomic landscape of SARS-CoV-2 variants isolated in Saudi Arabia in a comparative in silico study.

SARS-CoV-2 has become one of the unprecedented global health challenge for human population. Genomic signature studies of SARS-CoV-2 reveals relation between geographical location of the isolates and genetic diversity. The present work is an in silico, cross sectional study aimed to determine the genetic heterogeneity of SARS-CoV-2 variants isolated in Saudi Arabia compared to the first isolated strain NC_045512 (reference sequence). Each sequence was aligned against the reference sequence using local alignment search tool (NCBI) Nucleotide-BLAST. A total of 58 SARS-CoV-2 genomes were isolated in KSA and retrieved from NCBI. Our study shows that KSA variants demonstrated homology ranging between 99.96 and 99.98 % compared to the reference strain. There are 89 nucleotide changes that have been identified among the KSA variants; the most common nucleotide change was C: T accounting for 50.6% (45/89). These nucleotides changes resulted in 53.9% (48/89) missense mutations and 42.7% (38/89) silent mutations; while the majority of mutations- 48.3% (43/89) occurred in ORF1ab gene. All structural genes displayed mutations; N gene harbored 16.9% (15/89) mutations, S gene displayed 15.7% (14/89) mutations, M gene exhibited 2.2% (2/89) mutations and E gene showed only 1 mutation which was silent. The most frequently changed nucleotide was C3037T (silent mutation) and A23403G (D614G), each of which occurred in 57 variants out of 58 followed by C14408T (P4715L) and C241T (5′UTR) which were found in 56 and 55 variants respectively. The Phylogenetic trees showed that SARS-CoV-2 variants isolated in Saudi Arabia clustered together closely.

Identification of the novel KIR3DL1*00702 allele in a Northern Chinese Han individual.

The novel KIR3DL1*00702 allele differs from the closest allele KIR3DL1*00701 by a single silent mutation.