missense mutations
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Fuxiao Liu ◽  
Jiahui Lin ◽  
Qianqian Wang ◽  
Youming Zhang ◽  
Hu Shan

Canine distemper and canine parvoviral enteritis are infections caused by the canine distemper virus (CDV) and canine parvovirus type 2 (CPV-2), respectively. They are two common infectious diseases that cause high morbidity and mortality in affected dogs. Combination vaccines have been broadly used to protect dogs from infections of CDV, CPV-2, and other viruses. VP2 is the most abundant protein of the CPV-2 capsid. It elicits potent immunity in animals and, therefore, is widely used for designing subunit antigen-based vaccines. In this study, we rescued a recombinant CDV (QN vaccine strain) using reverse genetics. The recombinant CDV (rCDV-VP2) was demonstrated to express stably the VP2 in cells for at least 33 serial passages in vitro. Unfortunately, a nonsense mutation was initially identified in the VP2 open reading frame (ORF) at passage-34 (P34) and gradually became predominant in rCDV-VP2 quasispecies with passaging. Neither test strip detection nor indirect immunofluorescence assay demonstrated the expression of the VP2 at P50. The P50 rCDV-VP2 was subjected to next-generation sequencing, which totally identified 17 single-nucleotide variations (SNVs), consisting of 11 transitions and 6 transversions. Out of the 17 SNVs, 1 and 9 were identified as nonsense and missense mutations, respectively. Since the nonsense mutation arose in the VP2 ORF as early as P34, an earlier rCDV-VP2 progeny should be selected for the vaccination of animals in future experiments.

2022 ◽  
Vol 12 ◽  
Tannaz Moeini Shad ◽  
Reza Yazdani ◽  
Parisa Amirifar ◽  
Samaneh Delavari ◽  
Marzieh Heidarzadeh Arani ◽  

Ataxia-telangiectasia (AT) is a rare autosomal recessive neurodegenerative multisystem disorder. A minority of AT patients can present late-onset atypical presentations due to unknown mechanisms. The demographic, clinical, immunological and genetic data were collected by direct interview and examining the Iranian AT patients with late-onset manifestations. We also conducted a systematic literature review for reported atypical AT patients. We identified three Iranian AT patients (3/249, 1.2% of total registry) with later age at ataxia onset and slower neurologic progression despite elevated alpha-fetoprotein levels, history of respiratory infections, and immunological features of the syndrome. Of note, all patients developed autoimmunity in which a decrease of naïve T cells and regulatory T cells were observed. The literature searches also summarized data from 73 variant AT patients with atypical presentation indicating biallelic mild mutations mainly lead to an atypical phenotype with an increased risk of cancer. Variant AT patients present with milder phenotype or atypical form of classical symptoms causing under- or mis- diagnosis. Although missense mutations are more frequent, an atypical presentation can be associated with deleterious mutations due to unknown modifying factors.

Blood ◽  
2022 ◽  
Leif Ludwig ◽  
Caleb A Lareau ◽  
Erik L. Bao ◽  
Nan Liu ◽  
Taiju Utsugisawa ◽  

Master regulators, such as the hematopoietic transcription factor (TF) GATA1, play an essential role in orchestrating lineage commitment and differentiation. However, the precise mechanisms by which such TFs regulate transcription through interactions with specific cis-regulatory elements remain incompletely understood. Here, we describe a form of congenital hemolytic anemia caused by missense mutations in an intrinsically disordered region of GATA1, with a poorly understood role in transcriptional regulation. Through integrative functional approaches, we demonstrate that these mutations perturb GATA1 transcriptional activity by partially impairing nuclear localization and selectively altering precise chromatin occupancy by GATA1. These alterations in chromatin occupancy and concordant chromatin accessibility changes alter faithful gene expression, with failure to both effectively silence and activate select genes necessary for effective terminal red cell production. We demonstrate how disease-causing mutations can reveal regulatory mechanisms that enable the faithful genomic targeting of master TFs during cellular differentiation.

2022 ◽  
Vol 23 (2) ◽  
pp. 858
Sali Anies ◽  
Vincent Jallu ◽  
Julien Diharce ◽  
Tarun J. Narwani ◽  
Alexandre G. de Brevern

Integrin αIIbβ3, a glycoprotein complex expressed at the platelet surface, is involved in platelet aggregation and contributes to primary haemostasis. Several integrin αIIbβ3 polymorphisms prevent the aggregation that causes haemorrhagic syndromes, such as Glanzmann thrombasthenia (GT). Access to 3D structure allows understanding the structural effects of polymorphisms related to GT. In a previous analysis using Molecular Dynamics (MD) simulations of αIIb Calf-1 domain structure, it was observed that GT associated with single amino acid variation affects distant loops, but not the mutated position. In this study, experiments are extended to Calf-1, Thigh, and Calf-2 domains. Two loops in Calf-2 are unstructured and therefore are modelled expertly using biophysical restraints. Surprisingly, MD revealed the presence of rigid zones in these loops. Detailed analysis with structural alphabet, the Proteins Blocks (PBs), allowed observing local changes in highly flexible regions. The variant P741R located at C-terminal of Calf-1 revealed that the Calf-2 presence did not affect the results obtained with isolated Calf-1 domain. Simulations for Calf- 1+ Calf-2, and Thigh + Calf-1 variant systems are designed to comprehend the impact of five single amino acid variations in these domains. Distant conformational changes are observed, thus highlighting the potential role of allostery in the structural basis of GT.

PeerJ ◽  
2022 ◽  
Vol 10 ◽  
pp. e12750
Supharada Tessiri ◽  
Anchalee Techasen ◽  
Sarinya Kongpetch ◽  
Achira Namjan ◽  
Watcharin Loilome ◽  

Background Genetic alterations in ARID1A were detected at a high frequency in cholangiocarcinoma (CCA). Growing evidence indicates that the loss of ARID1A expression leads to activation of the PI3K/AKT pathway and increasing sensitivity of ARID1A-deficient cells for treatment with the PI3K/AKT inhibitor. Therefore, we investigated the association between genetic alterations of ARID1A and the PI3K/AKT pathway and evaluated the effect of AKT inhibition on ARID1A-deficient CCA cells. Methods Alterations of ARID1A, PI3K/AKT pathway-related genes, clinicopathological data and overall survival of 795 CCA patients were retrieved from cBio Cancer Genomics Portal (cBioPortal) databases. The association between genetic alterations and clinical data were analyzed. The effect of the AKT inhibitor (MK-2206) on ARID1A-deficient CCA cell lines and stable ARID1A-knockdown cell lines was investigated. Cell viability, apoptosis, and expression of AKT signaling were analyzed using an MTT assay, flow cytometry, and Western blots, respectively. Results The analysis of a total of 795 CCA samples revealed that ARID1A alterations significantly co-occurred with mutations of EPHA2 (p < 0.001), PIK3CA (p = 0.047), and LAMA1 (p = 0.024). Among the EPHA2 mutant CCA tumors, 82% of EPHA2 mutant tumors co-occurred with ARID1A truncating mutations. CCA tumors with ARID1A and EPHA2 mutations correlated with better survival compared to tumors with ARID1A mutations alone. We detected that 30% of patients with PIK3CA driver missense mutations harbored ARID1A-truncated mutations and 60% of LAMA1-mutated CCA co-occurred with truncating mutations of ARID1A. Interestingly, ARID1A-deficient CCA cell lines and ARID1A-knockdown CCA cells led to increased sensitivity to treatment with MK-2206 compared to the control. Treatment with MK-2206 induced apoptosis in ARID1A-knockdown KKU-213A and HUCCT1 cell lines and decreased the expression of pAKTS473 and mTOR. Conclusion These findings suggest a dependency of ARID1A-deficient CCA tumors with the activation of the PI3K/AKT-pathway, and that they may be more vulnerable to selective AKT pathway inhibitors which can be used therapeutically.

2022 ◽  
Vol 22 (1) ◽  
Tianchang Tao ◽  
Xianfen Meng ◽  
Ningda Xu ◽  
Jiarui Li ◽  
Yong Cheng ◽  

Abstract Background Retinopathy of prematurity (ROP) is a multifactorial retinal disease, involving both environmental and genetic factors; The purpose of this study is to evaluate the clinical presentations and genetic variants in Chinese patients with ROP. Methods A total of 36 patients diagnosed with ROP were enrolled in this study, their medical and ophthalmic histories were obtained, and comprehensive clinical examinations were performed. Genomic DNA was isolated from peripheral blood of ROP patients, polymerase chain reaction and direct sequencing of the associated pathogenic genes (FZD4, TSPAN12, and NDP) were performed. Results All patients exhibited the clinical manifestations of ROP. No mutations were detected in the TSPAN12 and NDP genes in all patients; Interestingly, three novel missense mutations were identified in the FZD4 gene (p.A2P, p.L79M, and p.Y378C) in four patients, for a detection rate of 11.1% (4/36). Conclusions This study expands the genotypic spectrum of FZD4 gene in ROP patients, and our findings underscore the importance of obtaining molecular analyses and comprehensive health screening for this retinal disease.

2022 ◽  
Michaela Fenckova ◽  
Villo Muha ◽  
Daniel Mariyappa ◽  
Marica Catinozzi ◽  
Ignacy Czajewski ◽  

O-GlcNAcylation is a reversible co-/post-translational modification involved in a multitude of cellular processes. The addition and removal of O-GlcNAc modification is controlled by two conserved enzymes, O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Mutations in OGT have recently been discovered to cause a novel Congenital Disorder of Glycosylation (OGT-CDG) that is characterized by intellectual disability. The mechanisms by which OGT-CDG mutations affect cognition remain unclear. We manipulated O-GlcNAc transferase and O-GlcNAc hydrolase activity in Drosophila and demonstrate an important role of O-GlcNAcylation in habituation learning and synaptic development at the larval neuromuscular junction. Introduction of patient-specific missense mutations into Drosophila O-GlcNAc transferase using CRISPR/Cas9 gene editing, leads to deficits in locomotor function and habituation learning. The habituation deficit can be corrected by blocking O-GlcNAc hydrolysis, indicating that OGT-CDG mutations affect cognitive function via reduced protein O-GlcNAcylation. This study establishes a critical role for O-GlcNAc cycling and disrupted O-GlcNAc transferase activity in cognitive dysfunction. These findings suggest that blocking O-GlcNAc hydrolysis is a potential treatment strategy for OGT-CDG.

Murat Sayan ◽  
Ayse Arikan ◽  
Murat Isbilen

Aims: This study determined SARS-CoV-2 variations by phylogenetic and virtual phenotyping analyses. Materials & methods: Strains isolated from 143 COVID-19 cases in Turkey in April 2021 were assessed. Illumina NexteraXT library preparation kits were processed for next-generation ]sequencing. Phylogenetic (neighbor-joining method) and virtual phenotyping analyses (Coronavirus Antiviral and Resistance Database [CoV-RDB] by Stanford University) were used for variant analysis. Results: B.1.1.7–1/2 (n = 103, 72%), B.1.351 (n = 5, 3%) and B.1.525 (n = 1, 1%) were identified among 109 SARS-CoV-2 variations by phylogenetic analysis and B.1.1.7 (n = 95, 66%), B.1.351 (n = 5, 4%), B.1.617 (n = 4, 3%), B.1.525 (n = 2, 1.4%), B.1.526-1 (n = 1, 0.6%) and missense mutations (n = 15, 10%) were reported by CoV-RDB. The two methods were 85% compatible and B.1.1.7 (alpha) was the most frequent SARS-CoV-2 variation in Turkey in April 2021. Conclusion: The Stanford CoV-RDB analysis method appears useful for SARS-CoV-2 lineage surveillance.

2022 ◽  
Cody A Ramirez ◽  
Felix Frenkel ◽  
Michelle Becker ◽  
Erica K Barnell ◽  
Ethan D McClain ◽  

Personalized cancer vaccines designed to target neoantigens represent a promising new treatment paradigm in oncology. In contrast to classical idiotype vaccines, we hypothesized that polyvalent vaccines could be engineered for the personalized treatment of follicular lymphoma (FL) using neoantigen discovery by combined whole exome sequencing (WES) and RNA sequencing (RNA-Seq). Fifty-eight tumor samples from 57 patients with FL underwent WES and RNA-Seq. Somatic and B-cell clonotype neoantigens were predicted and filtered to identify high-quality neoantigens. B-cell clonality was determined by alignment of B-cell receptor (BCR) CDR3 regions from RNA-Seq data, grouping at the protein level, and comparison to the BCR repertoire of RNA-Seq data from healthy individuals. An average of 52 somatic mutations per patient (range: 2-172) were identified, and two or more (median: 15) high-quality neoantigens were predicted for 56 of 58 samples. The predicted neoantigen peptides were composed of missense mutations (76%), indels (9%), gene fusions (3%), and BCR sequences (11%). Building off of these preclinical analyses, we initiated a pilot clinical trial using personalized neoantigen vaccination combined with PD-1 blockade in patients with relapsed or refractory FL (#NCT03121677). Synthetic long peptide (SLP) vaccines were successfully synthesized for and administered to all four patients enrolled to date. Initial results demonstrate feasibility, safety, and potential immunologic and clinical responses. Our study suggests that a genomics-driven personalized cancer vaccine strategy is feasible for patients with FL, and this may overcome prior challenges in the field.

2022 ◽  
Vol 13 (1) ◽  
M. S. Oud ◽  
R. M. Smits ◽  
H. E. Smith ◽  
F. K. Mastrorosa ◽  
G. S. Holt ◽  

AbstractDe novo mutations are known to play a prominent role in sporadic disorders with reduced fitness. We hypothesize that de novo mutations play an important role in severe male infertility and explain a portion of the genetic causes of this understudied disorder. To test this hypothesis, we utilize trio-based exome sequencing in a cohort of 185 infertile males and their unaffected parents. Following a systematic analysis, 29 of 145 rare (MAF < 0.1%) protein-altering de novo mutations are classified as possibly causative of the male infertility phenotype. We observed a significant enrichment of loss-of-function de novo mutations in loss-of-function-intolerant genes (p-value = 1.00 × 10−5) in infertile men compared to controls. Additionally, we detected a significant increase in predicted pathogenic de novo missense mutations affecting missense-intolerant genes (p-value = 5.01 × 10−4) in contrast to predicted benign de novo mutations. One gene we identify, RBM5, is an essential regulator of male germ cell pre-mRNA splicing and has been previously implicated in male infertility in mice. In a follow-up study, 6 rare pathogenic missense mutations affecting this gene are observed in a cohort of 2,506 infertile patients, whilst we find no such mutations in a cohort of 5,784 fertile men (p-value = 0.03). Our results provide evidence for the role of de novo mutations in severe male infertility and point to new candidate genes affecting fertility.

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