ARTICLE | doi:10.20944/preprints202005.0396.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; COVID-19; coronavirus; variant analysis; phylogenetic analysis; viral evolution
Online: 24 May 2020 (18:25:47 CEST)
The severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) viral genome is an RNA virus consisting of approximately 30,000 bases. As part of testing efforts, whole genome sequencing of human isolates has resulted in over 1,600 complete genomes publicly available from GenBank. We have performed a comparative phylogenetic analysis of the sequences, in order to detect common mutations within the population. Analysis of variants occurring within the assembled genomes yields 417 variants occurring in at least 1% of the completed genomes, including 229 within the 5’ untranslated region (UTR), 152 within the 3’UTR, 2 within intergenic regions and 34 within coding sequences.
ARTICLE | doi:10.20944/preprints202101.0473.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: molecular methods; virus; pandemic; Coronaviridae; COVID-19, diagnosis; UK variant; B.1.1.7 variant; 501Y.V2 variant
Online: 25 January 2021 (10:32:38 CET)
Starting from early 2020, the COVID-19 pandemic has caused a morbidity and mortality excess worldwide. Containment strategies rely firstly on rapid and sensitive laboratory diagnosis with molecular detection of the viral genome in respiratory samples being the gold standard. Reliability of diagnostic protocols could be affected by SARS-CoV-2 genetic variability. In fact, mutations occurring during SARS-CoV-2 genomic evolution can involve the regions targeted by the diagnostic probes. Following a review of the literature and an in silico analysis of the most recently described virus variants (including the UK B 1.1.7 and the South Africa 501Y.V2 variants), we conclude that the described genetic variability should have minimal or no effect on the sensitivity of existing diagnostic protocols for SARS-CoV-2 genome detection. However, given the continuous emergence of new variants, the situation should be monitored in the future, and protocols including multiple targets should be preferred.
ARTICLE | doi:10.20944/preprints202203.0155.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; Omicron variant; Monoclonal antibody; Neutralization; Spike protein
Online: 10 March 2022 (14:30:25 CET)
SARS-CoV-2 Omicron variants contain many mutations in its spike receptor binding domain, the target of all authorized monoclonal antibodies (mAbs). Determining the extent to which Omicron variants reduced mAb susceptibility is critical to preventing and treating COVID-19. We systematically reviewed PubMed and three preprint servers, last updated February 22, 2022, of the in vitro activity of authorized mAbs against the Omicron variants. Thirty-three studies were eligible including 33 containing Omicron BA.1 susceptibility data and five that also contained Omicron BA.2 susceptibility data. The first two authorized mAb combinations, bamlanivimab/etesevimab and casirivimab/imdevimab, were inactive against the Omicron BA.1 and BA.2 variants. In 24 studies, sotrovimab (third authorized mAb) displayed a median 4.1-fold (IQR: 2.4-7.6) reduced activity against Omicron BA.1 and, in four studies, a median 26-fold (IQR:16-35) reduced activity against Omicron BA.2. In 18 studies, cilgavimab and tixagevimab independently displayed median reductions in activity of >300-fold against Omicron BA.1, while in ten studies, the cilgavimab/tixagevimab combination (fourth authorized mAb preparation) displayed a median 63-fold (IQR: 26-145) reduced activity against Omicron BA.1. In two studies, cilgavimab was approximately 100-fold more susceptible to BA.2 than to BA.1. In two studies, bebtelovimab, the most recently authorized mAb, was fully active against both the Omicron variants. Disparate results between assays were common as evidenced by a median 42-fold range (IQR: 25-625) in IC50 between assays for the eight authorized individual mAbs and three authorized mAb combinations. Highly disparate results between published assays indicates a need for improved mAb susceptibility test standardization or inter-assay calibration.
ARTICLE | doi:10.20944/preprints202008.0206.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: tongue microbiome; salivary microbiome; amplicon sequence variant (ASV); operational taxonomical unit (OTU); denoising; DADA2; taxonomic classifier
Online: 8 August 2020 (09:29:46 CEST)
The bacterial composition of oral samples has traditionally been determined by PCR amplicon sequencing of 16S rRNA genes. Recent amplicon sequence variant (ASV)-based analyses of 16S rRNA genes differ from that based on operational taxonomic unit (OTU) clustering in the way it deals with sequences having potential errors. However, little information is available on its application in oral microbiome studies. Here, we conducted ASV-based analysis of oral microbiome samples using QIIME 2. We investigated the optimal parameters for sequence denoising, using DADA2, and found the trimming of the first 20 nucleotides from 5′-end of both paired reads avoided excessive sequence loss during chimera removal. Truncating reads at positions 240–245 allowed the removal of low-quality sequences while maintaining sufficient length to merge matching paired ends. Taxonomic assignment, using the naïve Bayes classifier trained with the V3-V4 region of reference 16S rRNA sequences in the extended human oral microbiome database (eHOMD), resulted in bacterial compositions similar to those of OTU-based analyses. Contrary to OTU-based clustering, ASV-based analysis showed taxonomic abundance at the genus or species level to not differ significantly in tongue microbiomes, regardless of brushing. QIIME 2 can, therefore, be a standard pipeline for ASV-based analysis of oral microbiomes.
ARTICLE | doi:10.20944/preprints202209.0390.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; COVID-19; asymptomatic; seroprevalence; Delta variant; Omicron variant; vaccination
Online: 26 September 2022 (09:41:01 CEST)
A significant proportion of SARS-CoV-2 infections in Africa are identified as asymptomatic, facilitating the silent spread of the virus especially in populated urban cities. With the surge of the highly transmissible Omicron variant, the inclusion of asymptomatics in epidemiological surveys is key in estimating true infections and seroprevalence in the population. The aim of the study was to determine seroprevalence, active infection and circulating variants in Accra, the capital city of Ghana during the Omicron wave. The study was a cross-sectional survey conducted in 22 municipalities in December 2021. Naso-oropharyngeal swabs and serum samples were collected from 1027 individuals aged 5 years and above, for detection of infection by RT-qPCR and estimation of total antibodies using the WANTAI ELISA kit. Our results show 10% SARS-CoV-2 prevalence, with the Omicron and Delta variants accounting for 44.1% and 8.8% of infections, respectively. Omicron was most prevalent (48.9.%) among the 20–39-year-olds. Asymptomatic individuals accounted for 75.2% of infections. Seropositivity within the population was 86.8%, with the 60+ year group having significantly higher likelihood of exposure (OR 10.22: 95% CI: 3.51-29.73; p<0.001). This high seroprevalence appears to have been as a result of increased vaccination among this group (OR 2.7: 95% CI 1.78-4.09, p < 0.001). The high seropositivity of SARS-CoV-2 in the capital could be a good indication of herd immunity among the population and while the low infection rate supports the role of vaccination in reducing viral transmission.
ARTICLE | doi:10.20944/preprints202012.0387.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: next-generation sequencing; database; variant annotation; variant classification; data management; clinical genomics
Online: 15 December 2020 (13:14:21 CET)
The rapid evolution of Next Generation Sequencing in clinical settings and the resulting challenge of variants interpretation in the light of constantly updated information, requires robust data management systems and organized approaches to variant reinterpretation. In this paper, we present iVar: a freely available and highly customizable tool provided with a user-friendly web interface. It represents a platform for the unified management of variants identified by different sequencing technologies. iVar accepts, as input, VCF files and text annotation files and elaborates them, optimizing data organization and avoiding redundancies. Updated annotations can be periodically re-uploaded and associated to variants as historicize attributes. Data can be visualized through variant-centered and sample-centered interfaces. A customizable search functionality can be exploited to periodically check if pathogenicity related data of a variant are changed over time. Patient recontacting ensuing from variant reinterpretation is made easier by iVar through the effective identification of all patients present in the database and carrying a specific variant. We tested iVar by uploading 4171 VCF files and 1463 annotation files, obtaining a database of 4166 samples and 22569 unique variants. iVar has proven to be a useful tool with good performances for collecting and managing data from medium-throughput
ARTICLE | doi:10.20944/preprints202205.0292.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Meier-Gorlin syndrome; Jeune syndrome; ORC6; exon skipping variant; prenatal genetic testing
Online: 23 May 2022 (10:18:24 CEST)
Meier–Gorlin syndrome (MGS) is a rare genetic developmental disorder that causes primordial proportional dwarfism, microtia, absent or hypoplastic patellae and other skeletal anomalies. Overlapping skeletal symptoms make MGS difficult to diagnose clinically. We describe a 3-year-old boy with short stature, recurrent respiratory infections, short-rib dysplasia, tower head and facial dysmorphisms who was admitted to the Tomsk Genetic Clinic to verify a clinical diagnosis of Jeune syndrome. Clinical Exome sequencing revealed two variants (compound heterozygosity) in the ORC6 gene: c.2T>C(p.Met1Thr) and c.449+5G>A. In silico analysis showed the pathogenicity of these two mutations and predicted a decrease in donor splicing site strength for c.449+5G>A. An in vitro minigene assay demonstrated that variant c.449+5G>A causes a complete skipping of exon 4 in ORC6 gene. The parents asked for urgent prenatal testing for MGS for the next pregnancy, but it ended in a miscarriage. Thus, this case report may help to prevent MGS misdiagnosis in the future. We also performed in silico and functional analyses of ORC6 mutations and developed a restriction fragment length polymorphism and haplotype-based short-tandem-repeat assay for prenatal genetic testing for MGS. These findings should elucidate MGS aetiology and improve the quality of genetic counselling for affected families.
ARTICLE | doi:10.20944/preprints202208.0209.v1
Online: 11 August 2022 (06:01:14 CEST)
Viral variant analysis is a bedrock of the disease surveillance. When combined with temporospatial analysis variant analysis can further the knowledge of disease spread in a study area. This paper suggests a method to perform the analysis in an operational setting which will allow for real-time surveillance of viral variants and allow local public health professionals to rapidly respond to changes in the evolution of the disease. This method includes three main subprocesses: preprocessing, analysis, and rendering. This method can be performed across multiple software platforms. A use case is given in which it was found that this method helped a hospital system understand the spread of SARS-CoV-2 in Northeast, Ohio.
ARTICLE | doi:10.20944/preprints202104.0401.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: Colorectal cancer; personalized medicine; biomarker; variant
Online: 15 April 2021 (08:10:42 CEST)
Discovery of novel variants from data derived from local population provides confident targets for developing biomarkers for personalized medicine. Biomarker discovery would be an important tool in advancing and utilizing the concept of precision and personalized medicine in the clinic. We identified the need to generate high quality sequencing data from local population and understand the pattern of occurrence of variants in colorectal cancer patients. In this report, we used archived samples from Saudi Arabia and used Ampliseq Comprehensive Cancer panel to identify novel somatic variants. We report a comprehensive analysis of next generation sequencing results with a coverage of >300X. We identified 466 novel variants which were previously unreported in COSMIC and ICGC databases. We analyzed the genes associated with these variants in terms of their frequency of occurrence, probable pathogenicity and clinicopathological features. Among pathogenic somatic variants, 174 were identified for the first time in large intestine. APC, RET and EGFR genes were most frequently mutated. Higher number of variants were identified in left colon. Occurrence of variants in ERBB2 was significantly correlated with those of EGFR and ATR genes. Network analyses of the identified genes provide functional perspective of the identified genes and suggest affected pathways and probable biomarker candidates. This report lays the ground work for biomarker discovery and identification of driver gene mutations in local population.
REVIEW | doi:10.20944/preprints201911.0085.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: mendelian disease; diagnostics; variant interpretation; variant prioritization; rna splicing; bioinformatics; machine learning; genomic medicine; effect prediction
Online: 8 November 2019 (04:07:16 CET)
Defects in pre-mRNA splicing are frequently a cause of Mendelian disease. Despite the advent of next-generation sequencing, allowing a deeper insight into a patient’s variant landscape, the ability to characterize variants causing splicing defects has not progressed with the same speed. To address this, recent years have seen a sharp spike in the number of splice prediction tools leveraging machine learning approaches, leaving clinical geneticists with a plethora of choices for in silico analysis. In this Review, some basic principles of machine learning are introduced in the context of genomics and splicing analysis. A critical comparative approach is then used to describe seven recent machine learning-based splice prediction tools, revealing highly diverse approaches and common caveats. We find that, although great progress has been made in producing specific and sensitive tools, there is still much scope for personalized approaches to prediction of variant impact on splicing. Such approaches may increase diagnostic yields and underpin improvements to patient care.
ARTICLE | doi:10.20944/preprints202007.0735.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Variant of Unknown Significance (VUS); Single-Nucleotide Variant (SNV); Variant Effect Prediction (VEP); Stacked Ensemble of Supervised Deep Learners (SESDL); Next Generation Sequencing (NGS); Alternative Allele Frequency (AAF).
Online: 31 July 2020 (06:13:53 CEST)
Pathogenicity is unknown for the majority of human gene variants. For prioritization of sequenced somatic and germline mutation variants, in silico approaches can be utilized. In this study, 84 million non-synonymous Single Nucleotide Variants (SNVs) in the human coding genome were annotated using consensus Variant Effect Prediction (cVEP) method. An algorithm, implemented as a stacked ensemble of supervised learners, performed combination of the 39 functional, conservation mutation impact scores from dbNSFP4.0. Adding gene indispensability score, accounting for differences in the pathogenicities of the variants in the essential and the mutation-tolerant genes, improved the predictions. For each SNV the consensus combination gives either a continuous-value pathogenicity score, or a categorical score in five classes: pathogenic, likely pathogenic, uncertain significance, likely benign, benign. The provided class database is aimed for direct use in clinical practice. The trained prediction models were 5-fold cross-validated on the evidence-based categorical annotations from the ClinVar database. The rankings of the scores based on their ability to predict pathogenicity were obtained. A two-step strategy using the rankings, scores and class annotations is suggested for filtering and prioritization of the human exome mutations in clinical and biological applications of NGS technology.
ARTICLE | doi:10.20944/preprints202301.0343.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Prostate cancer; Somatic point mutations; Copy number variation; Regulatory variant, Hi-C; Per-sonalized medicine; Biomedical machine learning
Online: 19 January 2023 (02:10:02 CET)
Prostate cancer (PC) is the most frequently diagnosed non-skin cancer in the world. Previous studies showed that genomic alterations represent the most common mechanism for molecular alterations that cause the development and progression of PC. Great efforts have been done to identify common protein-coding genetic variations; however, the impact of non-coding variations including regulatory genetic variants is not still well understood. To gain an understanding of the functional impact of genetic variants, particularly, regulatory variants in PC, we developed an integrative pipeline (AGV) that used whole genome/exome sequences, GWAS SNPs, chromosome conformation capture data, and ChIP-Seq signals to investigate the potential impact of genomic variants on the underlying target genes in PC. We identified 646 putative regulatory variants, of which 30 of them significantly altered the expression of at least one protein-coding gene. Our analysis of chromatin interactions data (Hi-C) revealed that the 30 putative regulatory variants may affect 131 coding and non-coding genes. Interestingly, our study showed the 131 protein-coding genes are involved in disease-related pathways including Reactome and MSigDB in which for most of them targeted treatment options are currently available. Together, our results provide a comprehensive map of genomic variants in PC and revealed their potential contribution to prostate cancer progression and development.
CASE REPORT | doi:10.20944/preprints202201.0177.v2
Subject: Medicine And Pharmacology, Cardiac And Cardiovascular Systems Keywords: variant effect maps; cardiac arrest; cardiovascular genetics
Online: 9 February 2023 (03:38:29 CET)
While genetic testing is becoming mainstream in the management of patients with potentially inherited cardiovascular disease, the prevalence of uncertain results severely limits its utility. One promising approach is to generate variant effect maps that report the function of all possible variants in a gene prospectively. The proactive clinical application of these maps is nascent, and it requires careful integration with current American College of Medical Genetics guidelines for variant interpretation. Here, we describe three pediatric cases of cardiac arrest or sudden cardiac death with variants of uncertain significance in calmodulin genes. We demonstrate the prospective clinical utility of a calmodulin variant effect map to inform variant interpretation, and therefore diagnosis and family care, in each case. This study was approved by the Stanford University and Vanderbilt University Medical Center IRBs. Consent was waived based on low risk of de-identified retrospective data collection per the IRB.
REVIEW | doi:10.20944/preprints202207.0051.v1
Subject: Medicine And Pharmacology, Pulmonary And Respiratory Medicine Keywords: SARS-CoV-2; Omicron; variant of concern
Online: 4 July 2022 (10:28:04 CEST)
For the first time in history, we have witnessed the origin and development of a pandemic. To handle the accelerated accumulation of viral mutations and to comprehend the virus' evolutionary adaptation in humans, an unparalleled program of genetic sequencing and monitoring of SARS-CoV-2 variants has been undertaken. Several scientists have theorized that, with the Omicron surge producing a more contagious but less severe disease, the end of COVID-19 is near. However, by analyzing the behavior shown by this virus for 2 years, we have noted that pandemic viruses do not always show a decreased virulence. Instead, it appears there is an evolutionary equilibrium between transmissibility and virulence. We have termed this concept “intermittent virulence”. The present work analyzes the temporal and epidemiological behavior of SARS-CoV-2 and suggests that there is a high possibility that new virulent variants will arise in the near future, although it is improbable that SARS-CoV-2´s virulence will be the same as was seen during the pandemic phase.
REVIEW | doi:10.20944/preprints201806.0191.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: rare disease; functional genomics; genetic variant validation
Online: 12 June 2018 (12:36:08 CEST)
Many insights into human disease have been built on experimental results in Drosophila, and research in fruit flies is often justified on the basis of its predictive value for questions related to human health. Additionally, there is now a growing recognition of the value of Drosophila for the study of rare human genetic diseases, either as a means of validating the causative nature of a candidate genetic variant found in patients, or as a means of obtaining functional information about a novel disease-linked gene when there is little known about it. For these reasons, funders in the US, Europe, and Canada have launched targeted programs to link human geneticists working on discovering new rare disease loci with researchers who work on the counterpart genes in Drosophila and other model organisms. Several of these initiatives are described here, as are a number of output publications that validate this new approach.
COMMUNICATION | doi:10.20944/preprints202310.1484.v1
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: COVID-19; SARS-CoV-2; SARS-CoV-2 Gamma variant; SARS-CoV-2 Delta variant; variant of concern (VOC); tropical and subtropical moist broadleaf forests (TSMF); vaccination; booster dose; lockdown; Italy
Online: 24 October 2023 (08:17:16 CEST)
Like all RNA viruses, SARS-CoV-2 shows high mutation rate, which has led to the emergence of new variants. Among them, Gamma and Delta developed at the turn of 2020-2021 in Amazonas and India, two ecoregions characterized by hot-humid weather, very similar to that of the summer season due to climate change in Italy, the first Western country to have been hit hard by COVID-19 and to experience lockdown restrictions in a democratic framework of 58,85 million people. Methods: during the state of national health emergency and the Draghi government, the Civil Defense Department daily released the aggregate data coming from the Ministry of Health, the Higher Institute of Health, the Independent Provinces and the Italian Regions, in order to inform about the pandemic situation in Italy. Among these data there were the number of deaths, hospitalizations in intensive care units (ICU), non-ICU patients, contagions, and performed swabs. By means of a team effort, we have collected and elaborated all these data, comparing COVID-19 pandemic in Italy during the summers 2020 (following the nationwide lockdown), 2021 and 2022. Results: from summer 2020 (before mass vaccination) to summers 2021 (after primary mass vaccination) and 2022 (after booster mass vaccination) all pandemic trend indicators have showed a sharp worsening in Italy. COVID-19 deaths increased by ≈298% and ≈834%, ICU hospitalizations by ≈386% and ≈310%, non-ICU hospitalizations by ≈224% and ≈600%, contagions by ≈627% and ≈6.850% (i.e. ≈68,50 times), swabs by ≈354% and ≈370%, and the mean positivity rate passed from ≈1% to ≈2% and ≈20%, respectively. Conclusions: SARS-CoV-2 can be transmitted in any climate, including areas with hot and humid weather, and the emergence of variants adapted to hot-humid climate may result in summer COVID-19 outbreaks, even in neither tropical nor subtropical countries. Although COVID-19 vaccines can confer cross-protection against newly emerging variants, this cross-immunity is obviously not absolute but limited, also taking into account that vaccine protection wanes considerably after 6 months. It follows that a subject vaccinated at the beginning of winter will not be completely covered in the height of summer, without forgetting the unvaccinated. As a final remark, the long nationwide lockdown made it possible to flatten SARS-CoV-2 circulation and, therefore, its negative impact on Italy during summer 2020.
ARTICLE | doi:10.20944/preprints202201.0333.v1
Subject: Computer Science And Mathematics, Artificial Intelligence And Machine Learning Keywords: Covid-19; Ensemble; Genome sequencing; Machine learning; Variant
Online: 21 January 2022 (15:17:58 CET)
Covid-19 has caused infections and deaths worldwide. While research in the field of Data Science has contributed good predictions of positive Covid-19 case numbers, this study's review of literature shows there is little research in the use of variants of the virus in predictions. We set out to define and evaluate novel variant features. We find that features relating to variant trends, thresholds and amino acid substitutions are especially powerful in two tasks. In the first task, predicting Covid-19 case numbers, accuracy improved from 71.53% without variant features to 82.12% with variant features. In the second task, predicting transmission severity of variants between two classes, we created a method to build some variable ensembles through selecting appropriate models that are generated with variant features. The test results showed that our ensembles are more accurate and reliable. One particular ensemble of 14 models correctly classified 90.91% of variants, outperforming other models including the popular Random Forest ensemble. In addition, as the variant features have represented more underlying information about Covid-19 pathophysiology, our ensemble methods use only a few data samples to achieve an accurate prediction. The ensemble of 14 models uses only 50 cases of each variant, an ability that could be exploited for early detection of highly infectious variants. These research findings may benefit public health professionals, policy makers, and the research community in the collective efforts to overcome this disease.
ARTICLE | doi:10.20944/preprints201807.0159.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: prostate cancer; HOXB13; benign prostatic hyperplasia; variant; Iran
Online: 10 July 2018 (05:36:05 CEST)
Background: Prostate cancer is a complex condition, in which both genetic and environmental factors concomitantly contribute to the tumor initiation and progression. Recently, HOXB13 has been proposed as a susceptibility gene for prostate cancer. Objective: So the current study was conducted to test the existence of potential variations in HOXB13 gene in Iranian men with prostate cancer (PCa) compared to benign prostatic hyperplasia (BPH) cases. Methods: The HOXB13 genetic status was screened in 51 samples, including 21 blood and tissue of PCa cases compared to 30 cases affected by BPH using PCR/sequencing. Then potential association between genomic DNA alterations in blood and tissue PCa specimens was investigated Results: Analysis of BPH tissues showed single nucleotide variations c.366C > T (rs) or c.513T > C (rs9900627) in exon 1, but not in exon 2. Evaluation of PCa tissues revealed two cases with both synonymous c.366C > T and c.513T > C variants and two cases with the synonymous c.366C > T variant in exon 1. The variants c.366C > T and c.513T > C simultaneously or separately were found blood samples of PCa patients. The novel variant c.127A > G in the exon 2 was detected in 1 PCa blood sample. Our analysis indicated a significant reciprocal correlation between HOXB13 mutation in tissue and blood in PCa cases (P=0.02). Conclusion: The variants in exon 2 of HOXB13 may influence the risk of prostate cancer. Also, evaluation of HOXB13 mutation may be considered as a novel marker for screening of PCa. Further investigations are warranted to evaluate the clinical significance of HOXB13 in Iranian population.
REVIEW | doi:10.20944/preprints202311.0482.v1
Subject: Biology And Life Sciences, Life Sciences Keywords: HHMS; AML; MDS; DDX41; TP53; SAMD9; SAMD9L; germline; variant
Online: 8 November 2023 (01:43:02 CET)
Due to the proliferation of genetic testing, pathogenic germline variants predisposing to hereditary hematological malignancy syndrome (HHMS) have been identified in an increasing number of genes. Consequently, the field of HHMS is gaining recognition among clinicians and scientists worldwide. Patients with germline genetic abnormalities often have poor outcomes and are candidates for allogeneic hematopoietic stem cell transplantation (HSCT). However, HSCT using blood from a related donor should be carefully considered because of the risk that the patient may inherit a pathogenic variant. At present, we now face the challenge of incorporating these advances into clinical practice for patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML) and optimizing the management and surveillance of patients and asymptomatic carriers, with the limitation that evidence-based guidelines are often inadequate. The 2016 revision of the WHO classification added a new section on myeloid malignant neoplasms, including MDS and AML with germline predisposition. The main syndromes can be classified into three groups. Those without pre-existing disease or organ dysfunction; DDX41, TP53, CEBPA, those with pre-existing platelet disorders; ANKRD26, ETV6, RUNX1, and those with other organ dysfunctions; SAMD9/SAMD9L, GATA2, and inherited bone marrow failure syndromes. In this review, we will outline the role of the genes involved in HHMS in order to clarify our understanding of HHMS.
ARTICLE | doi:10.20944/preprints202306.1363.v1
Subject: Biology And Life Sciences, Insect Science Keywords: Baculoviridae; bioinsecticide; genotypic variant; pathogenicity; virulence; occlusion body production
Online: 19 June 2023 (13:23:00 CEST)
Outbreaks of Anticarsia gemmatalis (Hübner, 1818) (Lepidoptera: Erebidae), a major pest of soybean, can be controlled below economic thresholds with methods that do not involve the application of synthetic insecticides. Formulations based on natural isolates of the Anticarsia gemmatalis multiple nucleopolyhedrovirus (AgMNPV) (Baculoviridae: Alphabaculovirus), played a significant role in integrated pest management programs in the early 2000s, but a new generation of chemical insecticides and transgenic soybean, have displaced AgMNPV-based products over the past decade. However, the marked genotypic variability present among and within alphabaculovirus isolates suggests that highly insecticidal genotypic variants can be isolated and used to reduce virus production costs or overcome isolate-dependent host resistance. This study aimed to select novel variants of AgMNPV with suitable insecticidal traits that could comple-ment the existing AgMNPV active ingredients. Three distinct AgMNPV isolates were compared by restriction endonuclease profile and in terms of their occlusion body (OB) pathogenicity. One isolate was selected (AgABB51) from which eighteen genotypic variants were plaque purified and characterized in terms of their insecticidal properties. The five most pathogenic variants varied in OB pathogenicity although none of them was faster-killing or had higher OB production characteristics than the wild-type isolate. We conclude that the AgABB51 wild-type isolates ap-pear to be genotypically structured for fast speed-of-kill and high OB production, both of which would favor transmission. Interactions among the component variants are likely to influence this insecticidal phenotype.
ARTICLE | doi:10.20944/preprints202304.0452.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: CD44; CD44 variant 3; monoclonal antibody; flow cytometry; immunohistochemistry
Online: 17 April 2023 (10:44:35 CEST)
Cluster of differentiation 44 (CD44) promotes tumor progression through the recruitment of growth factors, and the acquisition of stemness, invasiveness, and drug resistance. CD44 has multiple isoforms including CD44 standard (CD44s) and CD44 variants (CD44v), which have common and unique functions in tumor development. Therefore, the elucidation of each CD44 isoform’s function in tumor is essential for the establishment of CD44-targeting tumor therapy. We have established various anti-CD44s and anti-CD44v monoclonal antibodies (mAbs) through immunization of CD44v3–10-overexpressed cells. In this study, we established C44Mab-6 (IgG1, kappa), which recognized the CD44 variant 3-encoded region (CD44v3) determined by enzyme-linked immunosorbent assay. C44Mab-6 reacted with CD44v3–10-overexpressed Chinese hamster ovary (CHO)-K1 cells (CHO/CD44v3–10) or some cancer cell lines (COLO205 and HSC-3) by flow cytometry. The apparent KD of C44Mab-6 for CHO/CD44v3–10, COLO205, and HSC-3 was 1.5 × 10−9 M, 6.3 × 10−9 M, and 1.9 × 10−9 M, respectively. C44Mab-6 could detect the CD44v3–10 in western blotting, and stained the formalin-fixed paraffin-embedded tumor sections in immunohistochemistry. These results indicate that C44Mab-6 is useful for detecting CD44v3 in various experiments, and expected for the application of tumor diagnosis and therapy.
ARTICLE | doi:10.20944/preprints202301.0581.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: CD44; CD44 variant 5; monoclonal antibody; flow cytometry; immunohistochemistry
Online: 31 January 2023 (09:08:35 CET)
Pancreatic cancer exhibits a poor prognosis due to the lack of early diagnostic biomarkers and the resistance to conventional chemotherapy. CD44 has been known as a cancer stem cell marker, and plays tumor promotion and drug resistance in various cancers. Especially, the splicing variants are overexpressed in many carcinomas, and play essential roles in the cancer stemness, invasiveness or metastasis, and resistance to treatments. Therefore, the understanding of each CD44 variant (CD44v) function and distribution in carcinomas is essential for the establishment of CD44-targeting tumor therapy. In this study, we immunized mice with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells, and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-3; IgG1, kappa) recognized peptides of the variant 5-encoded region, indicating that C44Mab-3 is a specific mAb for CD44v5. Moreover, C44Mab-3 reacted with CHO/CD44v3–10 cells or pancreatic cancer cell lines (PK-1 and PK-8) by flow cytometry. The apparent KD of C44Mab-3 for CHO/CD44v3–10 and PK-1 was 7.1 × 10−10 M and 1.9 × 10−9 M, respectively. C44Mab-3 could detect the exogenous CD44v3–10 and endogenous CD44v5 in western blotting, and stained the formalin-fixed paraffin-embedded pancreatic cancer cells, but not normal pancreatic epithelial cells in immunohistochemistry. These results indicate that C44Mab-3 is useful for detecting CD44v5 in various applications, and expected for the application of pancreatic cancer diagnosis and therapy.
ARTICLE | doi:10.20944/preprints202301.0404.v1
Subject: Biology And Life Sciences, Virology Keywords: Omicron variant; Infectivity; Pathogenicity; Biothermodynamics; COVID-19; Viral evolution
Online: 23 January 2023 (08:53:40 CET)
The SARS-CoV-2 Hydra with many heads (variants) has been causing the COVID-19 pandemic for 3 years. The appearance of every new head (SARS-CoV-2 variant) causes a new pandemic wave. The last in the series is the XBB.1.5 “Kraken” variant. In the general public (social media) and in the scientific community (scientific journals), during the last several weeks since the variant has appeared, the question was raised of whether the infectivity of the new variant will be greater. This article attempts to provide the answer. Analysis of thermodynamic driving forces of binding and biosynthesis leads to the conclusion that infectivity of the XBB.1.5 variant could be increased to a certain extent. The pathogenicity of the XBB.1.5 variant seems to be unchanged compared to the other Omicron variants.
ARTICLE | doi:10.20944/preprints202212.0392.v1
Subject: Medicine And Pharmacology, Pathology And Pathobiology Keywords: CD44; CD44 variant 4; monoclonal antibody; flow cytometry; immunohistochemistry
Online: 21 December 2022 (07:40:21 CET)
CD44 has been known as a marker of tumor initiating cells, and plays pro-tumorigenic functions in many cancers. The splicing variants play critical roles in malignant progression of cancers by promoting the stemness, cancer cell invasion or metastasis, and resistance to chemo- and radiotherapy. To understand each CD44 variant (CD44v) function is essential to know the property of cancers and establishment of the therapy. However, the function of the variant 4-encoded region has not to be elucidated. Therefore, specific monoclonal antibodies (mAbs) against the variant 4 are indispensable for basic research, tumor diagnosis, and therapy. In this study, we established anti-CD44 variant 4 (CD44v4) monoclonal antibodies (mAbs) by immunizing mice with a peptide containing the variant 4-encoded region. We next performed flow cytometry, western blotting, and immunohistochemistry to characterized them. One of the established clones (C44Mab-108; IgG1, kappa) reacted with CD44v3-10-overexpressed Chinese hamster ovary-K1 cells (CHO/CD44v3-10). The KD of C44Mab-108 for CHO/CD44 v3-10 was 3.4 × 10−7 M. In western blot analysis, C44Mab-108 detected CD44v3-10 in the lysate of CHO/CD44v3-10 cells. Furthermore, C44Mab-108 stained formalin-fixed paraffin-embedded oral squamous carcinoma tissues in immunohistochemistry. These results indicated that C44Mab-108 is useful to detect CD44v4 in various applications.
BRIEF REPORT | doi:10.20944/preprints202205.0018.v2
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: SARS-CoV-2; Variant of Concern; BA.4; pandemic
Online: 6 May 2022 (11:55:20 CEST)
The rapid emergence and worldwide detection of the SARS-CoV-2 omicron variant underscore the importance of robust genomic surveillance systems and prompt information sharing among global public health partners. The Omicron variant has rapidly replaced the delta variant as a dominating SARS-CoV-2 variant because of natural selection, favoring the variant with higher infectivity and more strong vaccine breakthrough ability. Also known as B.1.1.529, Omicron has four sub-variants, BA.1, BA.2, BA.3, and BA.4. Among them, BA.1 is the currently prevailing sub-variant, BA.2 is found to be able to alarmingly re-infect patients initially infected by omicron BA.1. BA.3 sub-variants is a combination of mutations of BA.1 and BA.2, especially in the spike protein. Today emerging is the BA.4, herein described and first detected in Italy, harboring a new mutation, specifically a deletion in the ORF 1 ab gene, corresponding to KSF141_del in non-structural protein 1 (nsp1), a critical virulence factor able to suppress host translation. The bioinformatics comparison analysis with the other three sub-variants pointed out that the deletion was not present previously and was never reported until now. Therefore, we can speculate that omicron BA.4; will become a new dominating “variant of concern” and might also break vaccine protection . On the other hand, we show that other proteins are mutated in the BA.4 in particular, seven mutations are recognized in the nucleocapsid (N) protein, the capability of five different types of rapid antigenic tests to recognize it.
COMMUNICATION | doi:10.20944/preprints202203.0010.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; variant; Omicron; Delta; antigen; RT-qPCR
Online: 1 March 2022 (09:01:19 CET)
Rapid antigen detection tests (RAD) are commonly used for the diagnosis of SARS-CoV-2 infections. However, with the continuous emergence of new variants of concern (VOC) presenting various mutations potentially affecting the nucleocapsid protein, the analytical performances of these assays should be frequently reevaluated. One-hundred and twenty samples were selected and tested with both RT-qPCR and five commercial RAD commonly sold in Belgian pharmacies. Of these, direct whole genome sequencing identified the strains present in 116 samples, of which 70 were Delta and 46 were Omicron. Sensitivity across a wide range of Ct values (13.5 to 35.7; median = 21.3) were comparable and ranged from 70.0% to 77.1% for Delta strains and from 69.6% to 78.3% for Omicron strains. When taking swabs with a low viral load (Ct > 25), poor performances were observed for the Delta strains (20.0 to 40.0%) and, even more so, for Omicron strains (0.0 to 23.1%). Two devices failed to detect all samples (n = 13) containing Omicron strains with a low viral load. The poor performance observed with low viral loads is an important limitation of RAD, which is not sufficiently highlighted in the instruction for use of these devices.
ARTICLE | doi:10.20944/preprints202104.0643.v1
Subject: Engineering, Automotive Engineering Keywords: flexible rotating machinery; balancing method; speed-variant; acoustic feedback
Online: 23 April 2021 (13:18:55 CEST)
As rotary machines have become more complicated, balancing processes have been classified as a vital step in condition monitoring to ensure machines operate both reliably and safely. This is especially important for flexible machines which normally work at rotations speeds above critical limits. Imbalance is a common problem in flexible rotating machinery that can lead to extreme vibration and noise levels. This is one of the major reasons for studying various balancing methods applied to the vibration response of rotating machines. Recently, the relation between acoustic and vibration response during a rotary machine balancing process based on the Four-Run method has been presented for constant speed machines. This method cannot be applied to machines in start-up or shut-off. Hence, by considering the acoustic and vibration responses of a machine between its critical speeds, this research presents a new innovative speed-variant balancing method based on the original Four-Run method, named as "Peak to Peak for Critical Speeds (PPCS)". The proposed method consists of two major types of application: the first is in the Run-up of the machine and the second is in Shut-down. Experimental laboratory results show that the PPCS method can be implemented for speed-variant and flexible rotary machines during run-up or shut-down transient processes based on acoustic and vibration measurements. As a phase-less and a contactless method, the PPCS can be employed as an innovative and readily available method for condition monitoring in the future.
ARTICLE | doi:10.20944/preprints202103.0257.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: SLC15A4; germline variant; familial colorectal cancer; whole exome sequencing
Online: 9 March 2021 (10:24:33 CET)
About 15% of colorectal cancer (CRC) patients have first-degree relatives affected by the same malignancy. However, for most families the cause of familial aggregation of CRC is unknown. In order to identify novel high-to-moderate penetrant germline variants underlying CRC susceptibility, we performed whole exome sequencing (WES) on four CRC cases and two unaffected family members of a Polish family without any mutation in known CRC predisposition genes. After WES, we used our in-house developed Familial Cancer Variant Prioritization Pipeline and identified two novel variants in the solute carrier family 15 member 4 (SLC15A4) gene. The heterozygous missense variant, p. Y444C, was predicted to affect the phylogenetically conserved PTR2/POT domain and to have a deleterious effect on the function of the encoded peptide/histidine transporter. The other variant was located in the upstream region of the same gene (GRCh37.p13, 12_129308531_C_T; 43bp upstream of transcription start site, ENST00000266771.5) and it was annotated to affect the promoter region of SLC15A4 as well as binding sites of 17 different transcription factors. Our findings of two distinct variants in the same gene may indicate a synergistic up-regulation of SLC15A4 as the underlying genetic cause and implicate this gene for the first time in genetic inheritance of familial CRC.
ARTICLE | doi:10.20944/preprints202103.0121.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Familial colorectal cancer; SRC; germline variant; whole genome sequencing
Online: 3 March 2021 (09:52:06 CET)
Colorectal cancer (CRC) shows one of the largest proportions of familial cases among different malignancies, but only 5-10% of all CRC cases are linked to mutations in established predisposition genes. Thus, familial CRC constitutes a promising target for the identification of novel, high- to moderate-penetrance germline variants underlying cancer susceptibility by next generation sequencing. In this study, we performed whole genome sequencing on 3 members of a family with CRC aggregation. Subsequent integrative in silico analysis using our in-house developed variant prioritization pipeline resulted in the identification of a novel germline missense variant in SRC gene (V177M), a proto-oncogene highly upregulated in CRC. Functional validation experiments in HT-29 cells showed that introduction of SRCV177M resulted in increased cell proliferation and enhanced protein expression of phospho-SRC (Y419), a potential marker for SRC activity. Upregulation of paxillin, β-Catenin and STAT3 mRNA levels, increased levels of phospho-ERK, CREB and CCND1 proteins and downregulation of the tumor suppressor p53 further proposed the activation of several pathways due to the SRCV177M variant. The findings of our pedigree-based study contribute to the exploration of the genetic background of familial CRC and bring insights into the molecular basis of upregulated SRC activity and downstream pathways in colorectal carcinogenesis.
ARTICLE | doi:10.20944/preprints202009.0602.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: COVID-19; Genomes; microRNA; SARS-CoV-2; Variant discovery
Online: 25 September 2020 (10:14:46 CEST)
Background SARS-CoV-2 has generated a life-treating pandemic and is the main challenge of this century. Some untranslated regions (UTRs) in SARS-CoV-2 genome, specifically leader sequence and transcription regulatory sequence (TRS) in 5’UTR, can be considered as Achilles' heel of virus. Leader sequence are found at the 5' ends of all encoded transcripts that highlights its importance. TRS can explain the host range and pathogenicity of coronavirus. However, our knowledge on the evolution and the role of UTRs in SARS-CoV-2 pathogenicity is very limited. This study is a pioneering attempt to unravel the evolution of key regions in 5' UTR of SARS-CoV-2 and discover the inhibitory microRNAs against 5' UTR of virus. Methods Evolution of TRS and leader sequence was compared between human pathogenic (SARS-CoV-2, SARS, and MERS) and non-pathogenic (bovine) coronaviruses. Profiling of microRNAs that can inactive the key UTR regions of coronaviruses, UTR-inhibitory microRNAs, was carried out. Findings We found a distinguished pattern of evolution in leader sequence and TRS of SARS-CoV-2, compared to the other coronaviruses. Mining all available microRNA families against leader sequences of coronaviruses resulted in discovery of 39 microRNAs with an acceptable thermodynamic binding energy against SARS-COV-2, SARS, MERS, Bat Coronavirus, or Bovine Coronavirus. Multivariate analysis demonstrated a distinguished pattern of binding of leader sequence of SARS-CoV-2 against microRNAs, with a lower binding stability. hsa-MIR-5004-3p was the only human microRNA that can target leader sequence of SARS and SARS-CoV-2. However, its binding stability remarkably decreased in SARS-COV-2 (-19.4 kcal/mol), compared to SARS-COV-2 (-25.9 kcal/mol). We found an insertion-type mutation in leader sequence of SARS-COV-2 that results in lower binding stability and escaping of viral leader sequence from hsa-MIR-5004-3p. Altogether, we suggest lack of innate human inhibitory microRNAs to bind to leader sequence and TRS of SARS-CoV-2 contributes to its high replication in infected human cells. On the other hand, mining of two hundred million deposited human genomic variants led us to discovery of 49 missense and splice-disrupt mutations in genomic structure of hsa-MIR-5004-3p. These mutations can negatively affect hsa-MIR-5004-3p function in preventing SARS-CoV-2 replication. Interpretation This study unravels the evolution of key regions in 5’UTR of SARS-CoV-2. Inducing microRNAs to bind to the leader sequence and TRS regions by drugs or food supplements can reduce virus replication. Enhancing the microRNA defence machinery against TRS and leader of virus has a potential to prevent SARS-CoV-2 infection at the first place. The mentioned strategy is rapidly achievable against COVID-19. Missense variation in genomic sequence of 5’UTR inhibitory microRNAs, such as hsa-MIR-5004-3p, can be considered as risk factor of COVID-19.
CASE REPORT | doi:10.20944/preprints202008.0669.v1
Subject: Medicine And Pharmacology, Neuroscience And Neurology Keywords: Miller Fisher Syndrome; Guillain Barré Syndrome variant; Dysphonia; Dysphagia
Online: 30 August 2020 (14:41:29 CEST)
Miller Fisher Syndrome (MFS) is a rare variant of Guillain-Barré Syndrome (GBS). It is largely a clinical diagnosis based on the classical features of ataxia, areflexia, and opthalmoplegia. Its clinical evolution is most often favorable. However, other neurological signs and symptoms may also be present. Supportive laboratory studies (positivity of antibodies, CSF albumin-cytological dissociation and nerve conduction studies) are useful especially in uncommon presentations. We report a case of a 74-year-old patient who exhibited dysphonia and difficulty to swallowing previously to the classic triad of ataxia, areflexia, and opthalmoplegia, characteristic of MFS. CSF analysis demonstrates an albumin-cytological dissociation but anti-GQ1b antibody were negative. The patient has spontaneously and completely recovered after several weeks.
BRIEF REPORT | doi:10.20944/preprints202005.0466.v1
Subject: Biology And Life Sciences, Virology Keywords: COVID-19; SARS-CoV-2; variant; low pathogenicity; Singapore
Online: 29 May 2020 (12:47:07 CEST)
Number of confirmed cases of COVID-19 caused by SARS-CoV-2 exceeded 5 million as of May 21, 2020. Global average of the case fatality rate of COVID-19 is about 7% so far. There exist variations in case fatality rates among countries. Particularly, Singapore and Qatar have exceptionally low case fatality rates with 0.1% while France’s rate is almost 20%. Since no magic bullet treatment for COVID-19 exists, we investigated SARS-CoV-2 strains specific to Singapore in this study to identify a clade with low pathogenicity. Variant analysis revealed that a clade with variants ORF1ab L3606F, A4489V, S2015R, T2016K, and N P13L is common in Singapore. Based on our analysis of variants and historical case statistics, the clade is dominant in a recent surge. Therefore, we suggest that low case fatality rate of Singapore possibly is attributed to the clade. Although contribution of each variant to the low pathogenicity is not clear, L3606F alone does not accomplish such low pathogenicity from the comparison with case fatality data from Japan, where L3606F is dominant. Further investigation is necessary to conclude to validate this finding.
TECHNICAL NOTE | doi:10.20944/preprints202311.1130.v1
Subject: Computer Science And Mathematics, Software Keywords: Genomics; Variant Interpretation; Generative AI; Genome analysis; Rare Diseases; Bioinformatics
Online: 17 November 2023 (05:24:56 CET)
In the modern era of genomic research, the scientific community is witnessing an explosive growth in the volume of published findings. While this abundance of data offers invaluable insights, it also places a pressing responsibility on genetic professionals and researchers to stay informed about the latest findings and their clinical significance. Genomic variant interpretation is currently facing a challenge in identifying the most up-to-date and relevant scientific papers, while also extracting meaningful information to accelerate the process from clinical assessment to reporting. Computer-aided literature search and summarization can play a pivotal role in this context. By synthesizing complex genomic findings into concise, interpretable summaries, this approach facilitates the translation of extensive genomic datasets into clinically relevant insights. To bridge this gap, we present VarChat (varchat.engenome.com), an innovative tool based on generative AI, developed to find and summarize the fragmented scientific literature associated with genomic variants into brief yet informative texts.VarChat provides users with a concise description of specific genetic variants, detailing their impact on related proteins and possible effects on human health. Additionally, VarChat offers direct links to related scientific trustable sources, and encourages deeper research. Availability: VarChat is freely available at varchat.engenome.com
ARTICLE | doi:10.20944/preprints202307.0433.v1
Subject: Medicine And Pharmacology, Medicine And Pharmacology Keywords: PCNA; p15; FRET; inhibitor screening; ATLD2; disease; variant; stability; aggregation
Online: 6 July 2023 (11:53:02 CEST)
Proliferating cell nuclear antigen (PCNA) is the key regulator of human DNA metabolism. One important interaction partner is p15, involved in DNA replication and repair. Targeting the PCNA-p15 interaction is a promising therapeutic strategy against cancer. Here, a Förster resonance energy transfer (FRET)-based assay for the analysis of PCNA-p15 interaction was developed. Next to the application as screening tool for the identification and characterization of PCNA-p15 interaction inhibitors, the assay is also suitable for the investigation of mutation-induced changes in their affinity. This is particularly useful for analysing disease associated PCNA or p15 variants at the molecular level. Recently, the PCNA variant C148S has been associated with Ataxia-telangiectasia-like disorder type 2 (ATLD2). ATLD2 is a neurodegenerative disease based on defects in DNA repair due to an impaired PCNA. Incubation time dependent FRET measurements indicated no effect on PCNAC148S-p15 affinity, but on PCNA stability. The impaired stability and increased aggregation behaviour of PCNAC148S was confirmed by intrinsic tryptophan fluorescence, DSF and AF4 measurements. The analysis of the disease associated PCNA variant demonstrated the versatility of the interaction assay as developed.
ARTICLE | doi:10.20944/preprints202305.0995.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: CD44 variant 8; monoclonal antibody; gastric cancer; flow cytometry; immunohistochemistry
Online: 15 May 2023 (07:41:36 CEST)
Gastric cancer (GC) is the third leading cause of cancer-related deaths worldwide. GC with peritoneal metastasis exhibits a poor prognosis due to the lack of diagnostic biomarkers and effective therapy. A comprehensive analysis of malignant ascites identified the genomic alterations and significant amplifications of cancer driver genes, including CD44. CD44 and its splicing variants are overexpressed in tumors, and play crucial roles in the acquisition of invasiveness, stemness, and resistance to treatments. Therefore, the development of CD44-targeting monoclonal antibodies (mAbs) is important for GC diagnosis and therapy. In this study, we immunized mice with CD44v3–10-overexpressed PANC-1 cells and established several dozens of clones that produce anti-CD44v3–10 mAbs. One of the clones (C44Mab-94; IgG1, kappa) recognized the variant-8-encoded region and peptide, indicating that C44Mab-94 is a specific mAb for CD44v8. Furthermore, C44Mab-94 could recognize CHO/CD44v3–10 cells, oral squamous cell carcinoma cell line (HSC-3), or GC cell lines (MKN45 and NUGC-4) in flow cytometric analyses. C44Mab-94 could detect the exogenous CD44v3–10 and endogenous CD44v8 in western blotting and stained the formalin-fixed paraffin-embedded gastric cancer cells in immunohistochemistry. These results indicate that C44Mab-94 is useful for detecting CD44v8 in various applications and is expected to be useful for the application of GC diagnosis and therapy.
ARTICLE | doi:10.20944/preprints202305.0784.v1
Subject: Medicine And Pharmacology, Cardiac And Cardiovascular Systems Keywords: Arrhythmogenic Cardiomyopathy; PKP2; Founder pathogenic variant; Arrhythmic events; Heart Failure
Online: 11 May 2023 (04:38:37 CEST)
Introduction and objectives: arrhythmogenic cardiomyopathy (ACM) is a hereditary heart disease defined by the progressive replacement of the ventricular myocardium with fibroadipose tissue, which can act as a substrate for arrhythmias, sudden death, or even give rise to heart failure (HF). Sudden death is frequently the first manifestation of the disease, particularly among young patients. The aim of this study is to describe a new pathogenic variant in the PKP2 gene. Methods: A descriptive observational study that included 8 initially non-interrelated families with a diagnosis of ACM undergoing follow-up at our HF and Familial Cardiomyopathies Unit, who were carriers of the NM_004572.3:c.775_776insG; p. (Glu259Glyfs*77) variant in PKP2 gene. The genetic testing employed next-generation sequencing for the index cases, and the Sanger method for the targeted study with family members. We compiled personal and family histories, demographic and clinical characteristics, data from the additional tests at the time of diagnosis, and arrhythmic events at diagnosis and during follow-up. Results: We included 47 subjects, of whom 8 were index cases (17%). Among the evaluated family members, 16 (34%) were carriers of the genetic variant, 3 of whom also had a diagnosis of ACM. The majority were women (26 patients; 55.3%), with a mean age on diagnosis of 48.9 ± 18.6 years, and a median follow-up of 39 [24-59] months. Worthy of note are the high incidence of arrhythmic events as the form of presentation and in follow-up (21.5% and 20.9% respectively), and the onset of HF in 25% of the sample. The most frequent ventricular involvements were right (4 patients 16.7%) and biventricular (4 patients 16.7%); we found no statistical differences in any of the variables analysed. Conclusions: This variant is a pathogenic variant of gene PKP2 that has not previously been described and is not present in the control groups associated with ACM. It has incomplete penetrance, a highly variable phenotypic expressivity, and was identified in 8 families of our geographical area in Malaga (Andalusia, Spain), where we can establish a founder effect and describe the clinical and risk characteristics.
ARTICLE | doi:10.20944/preprints202302.0437.v1
Subject: Medicine And Pharmacology, Pathology And Pathobiology Keywords: CD44; CD44 variant 7/8; monoclonal antibody; flow cytometry; immunohistochemistry
Online: 27 February 2023 (04:13:02 CET)
Cluster of differentiation 44 (CD44) has been investigated as a cancer stem cell (CSC) marker, and plays critical roles in tumor malignant progression. The splicing variants are overexpressed in many carcinomas, especially squamous cell carcinomas, and play critical roles in the promotion of tumor metastasis, the acquisition of CSC properties, and resistance to treatments. Therefore, each CD44 variant (CD44v) function and distribution in carcinomas should be clarified for the establishment of novel tumor diagnosis and therapy. In this study, we immunized mice with a CD44 variant (CD44v3−10) ectodomain and established various anti-CD44 monoclonal antibodies (mAbs). One of the established clones (C44Mab-34; IgG1, kappa) recognized a peptide which covers both variant 7 and 8-encoded region, indicating that C44Mab-34 is a specific mAb for CD44v7/8. Moreover, C44Mab-34 reacted with CD44v3–10-overexpressed Chinese hamster ovary-K1 (CHO) cells or oral squamous cell carcinoma (OSCC) cell line (HSC-3) by flow cytometry. The apparent KD of C44Mab-34 for CHO/CD44v3–10 and HSC-3 was 1.4 × 10−9 M and 3.2 × 10−9 M, respectively. C44Mab-34 could detect CD44v3–10 in western blotting, and stained the formalin-fixed paraffin-embedded OSCC in immunohistochemistry. These results indicate that C44Mab-34 is useful for detecting CD44v7/8 in various applications, and expected for the application of OSCC diagnosis and therapy.
REVIEW | doi:10.20944/preprints202302.0132.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: Chromosomal instability; Cancer; Genome evolution; Speciation; Structural variant; Meiosis; micronuclei
Online: 7 February 2023 (12:14:31 CET)
The integrity of the genome is crucial for the survival of all living organisms. However, genomes need to adapt to survive certain pressures and for this propose use several mechanisms to diversify. Chromosomal instability (CIN) is one of the main mechanism leading to the creation of genomic heterogeneity by altering the number of chromosomes as well as changing their structures. In this review we will discuss the different chromosomal patterns and changes observed in speciation, in evolutional biology as well as during tumor progression. By nature, human genome shows induction of diversity during gametogenesis but as well during tumorigenesis that can conclude in drastic changes such as whole genome doubling to more discrete changes as indels as well as the recent discovered complex chromosomal rearrangement chromothripsis. More importantly, changes observed during speciation are strikingly similar to the genomic evolution observed during tumor progression and resistance to therapy. The different origins of CIN will be treated as the importance of double strand breaks (DSB) or the consequences of micronuclei. We will also explain the mechanisms behind the controlled DSBs and recombination of homologous chromosomes observed during meiosis, to explain how errors lead to similar pattern observed during tumorigenesis. Then, we will also list several diseases associated to CIN resulting in fertility issue, miscarriage, genetic rare diseases and cancer. Understanding better chromosomal instability as a whole is primordial for the understanding of mechanisms leading to tumor progression.
ARTICLE | doi:10.20944/preprints202301.0327.v1
Subject: Biology And Life Sciences, Virology Keywords: COVID-19; variant of concern; infectivity; pathogenicity; evolution; Gibbs energy
Online: 18 January 2023 (08:40:33 CET)
SARS-CoV-2 resembles the ancient mythical creature Hydra. Just like with the Hydra, when one head is cut, it is followed by appearance of two more heads, suppression of one SARS-CoV-2 variant causes appearance of newer variants. Unlike Hydra that grows identical heads, newer SARS-CoV-2 variants are usually more infective, which can be observed as time evolution of the virus at hand, which occurs through acquisition of mutations during time. The appearance of new variants is followed by appearance of new COVID-19 pandemic waves. With the appearance of new pandemic waves and determining of sequences, in the scientific community and general public the question is always raised of whether the new variant will be more virulent and more pathogenic. The two variants characterized in this paper, BA.5.2 and BF.7, have caused a pandemic wave during the late 2022. This paper gives full chemical and thermodynamic characterization of the BA.5.2 and BF.7 variants of SARS-CoV-2. Having in mind that Gibbs energy of binding and biosynthesis represent the driving forces for the viral life cycle, based on the calculated thermodynamic properties we can conclude that the newer variants are more infective than earlier ones, but that their pathogenicity has not changed.
ARTICLE | doi:10.20944/preprints202209.0362.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: RNA-Seq; Vitamin K; Comorbidities; Differential Expressed Genes; Variant analysis
Online: 23 September 2022 (09:13:29 CEST)
Systems genetics is key for integrating a large number of variants associated with diseases. Vitamin K (VK) is one of the scarcely studied conditions in lieu of ascertaining either the differentially expressed genes (DEGs) or variants in an individual subpopulation of diseased phenotypes associated with VK, viz. myocardial infarction, renal failure, prostate cancer, thrombosis, thrombocytopenia, coagulation related diseases to name a few. In this work, we have screened characteristic DEGs common to three VK-related diseases, viz. myocardial infarction, renal failure and prostate cancer and asked whether or not any DEGs in addition to pathogenic variants are common to these conditions. We attempt to bridge the gap in finding characteristic biomarkers and discuss the role of long noncoding RNAs (lncRNAs) in the biogenesis of VK deficiencies.
ARTICLE | doi:10.20944/preprints202205.0131.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; Variants of Concern; Delta Variant; genomic surveillance.
Online: 10 May 2022 (09:44:11 CEST)
In this study, we analyzed sequences of SARS-CoV-2 isolates of the Delta variant in Mexico, which completely replaced other previously circulating variants in the country due to its transmission advantage. Among Delta sublineages detected, 81.5 % were classified as AY.20, AY.26, and AY.100. According to publicly available data, these sublineages only reached a world prevalence of less than 1%, suggesting a possible Mexican origin. The signature mutations of these sublineages are described, and phylogenetic analyses and haplotype networks were used to track their spread across the country. Other frequently detected sublineages include AY.3, AY.62, AY.103, and AY.113. Over time, the principal sublineages showed different geographical distributions, with AY.20 predominant in Central Mexico, AY.26 in the North, and AY.100 in the Northwest and South/Southeast. This work describes the circulation, from May to November 2021, of the primary sublineages of the Delta variants associated to the third wave of the COVID-19 pandemic in Mexico and reinforces the importance of SARS-CoV-2 genomic surveillance for timely identification of emerging variants that may impact public health.
ARTICLE | doi:10.20944/preprints202204.0266.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS CoV-2; Variant of Concern; Omicron; mutation; genomic surveillance
Online: 28 April 2022 (03:28:50 CEST)
Genomic surveillance represents an important strategy for understanding evolutionary mechanisms, transmission profile, and infectivity of different SARS-CoV-2 variants. We assessed the epidemiological profile of 366 individuals who tested positive for SARS-CoV-2 from 29 municipalities in Rondônia between December 2021 to March 2022. Samples were collected, RNA was ex-tracted and screened using RT-qPCR for Alpha, Beta, Gamma, Delta and Omicron VOCs and viral quantification was performed. Sequences were analyzed for phylogeny, mutations and lineages. Of the samples analyzed, 93.71% were positive for the Omicron variant and 6.28% were positive for the Delta variant. The symptoms observed were cough, sore throat, and fever, with a mean duration of 5 days; no hospitalizations or deaths were reported. We noted that among the positive individuals, 51% had been immunized with two doses, 22% received three doses, 13% received one dose, and 13% were not immunized. Just 242 samples were amenable to analysis for alignment and phylogenetic characterization; corresponding to variants BA.1 and BA.1.1; a total of 120 mutations were identified, 36% of which were found in the S gene. In conclusion, there was a high frequency of mutations in the SARS-CoV-2 genome, but no record of clinical severity, demonstrating the positive effect of vaccination.
ARTICLE | doi:10.20944/preprints202203.0194.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: HLA; MHC class I; polymorphism; variant; type 2 diabetes; mexican
Online: 15 March 2022 (03:49:43 CET)
Type 2 diabetes has been linked to the expression of Human Leukocyte Antigens, principally to the major Histocompatibility Complex Class II and only scarce reports to Major Histocompatibility Complex class I in specific populations. The objective of the present work was to explore the presence of polymorphisms in the MHC class I related to Type 2 diabetes in the Mexican population using the GWAS SIGMA database. This database contains information of 3,848 Mexican individuals with type 2 diabetes and 4,366 control individuals from the same population without clinical or hereditary history of the disease. The searching criteria considered a P value < 0.005 and odds ratio, OR > 1.0. Ten novel statistically significant nucleotide variants were identified: four polymorphisms associated with HLA-A (A*03:01:01:01), and six with HLA-C (C*01:02:01:01). These alleles have a high prevalence in Latin American populations and could potentially be associated with autoimmunity mechanisms related with the development of Type 2 diabetes complications.
Subject: Biology And Life Sciences, Virology Keywords: ACE2; Spike protein; SARS-CoV2; death rate; polymorphism; isoform variant; CD157, sankramikogenomics
Online: 17 May 2020 (14:51:39 CEST)
The 2019-Novel Coronavirus has currently gripped the world in terror, affecting 210 countries and territories. Originating from Wuhan, Hubei province, China, the virus has spread so rapidly throughout the world and has already claimed 308,927 lives and is currently afflicting 4.6 million people. The US has over 1.48 million confirmed cases of COVID-19, followed by Spain, Italy, France, UK, Germany, Turkey, Russia, Iran, and China. On careful inspection of the COVID-19 statistics, a peculiar unsettling trend becomes apparent. Western European countries and the US appear to have difficulties in overcoming the catastrophe. In contrast, countries in East Asia, Middle East and mid-Europe have sorted out the situation. Here, we will highlight this trend and propose the importance of infection-genomics (sankramikogenomics), in understanding the susceptibility to COVID-19 and the severity of disease progress. More detailed, systematic evaluation may also identify more susceptible populations. We will also highlight mere 12-fold lower affinity is insufficient to ignore CD147, as interactions occur between tens of spike proteins and equal number of cell surface ACE2 and/or CD147. Thus, both receptors are important to understand sankramikogenomics and severity of COVID-19. The observed ethnic differences in COVID severities may be due to variations in structure or tissue-specific expression (alternate splicing and accessibility) of both the target receptors. Research on both receptors may help in designing improved therapeutic strategies to fight COVID-19. Similar to pharmacogenomics to drug development and precision medicine, Sankramikogenomics will become an important field in other infectious diseases and pathogenicity.
ARTICLE | doi:10.20944/preprints201811.0280.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: HSP47; missense mutation; mutational hotspot; variant analysis; cancer database; chaperone
Online: 12 November 2018 (10:23:29 CET)
Heat shock protein 47kDa (HSP47) serves as a client-specific chaperone, essential for collagen biosynthesis and its folding and structural assembly. To date, there is no comprehensive study on mutational hotspots and protein network for human HSP47. Using five different human mutational databases, we deduced a comprehensive list of human HSP47 mutations and we found 24 67, 50, 43 and 2 deleterious mutations from the 1000 genomes data, gnomAD, COSMICv86, cBioPortal, and CanVar. We identified thirteen top-ranked missense mutations of HSP47 with the stringent cut-off of CADD score (>25) and Grantham score (≥151) as Ser76Trp, Arg103Cys, Arg116Cys, Ser159Phe, Arg167Cys, Arg280Cys, Trp293Cys, Gly323Trp, Arg339Cys, Arg373Cys, Arg377Cys, Ser399Phe, and Arg405Cys with the arginine-cysteine change as the predominant mutation. We also found that HSP47 is up-regulated and down-regulated in 11 and 4 of cancers types. Upon constructing protein interactome map of human HSP47, we found that a set of molecular chaperones is interaction partners of HSP47, which included two copies each of CREB binding proteins, HSP27, HSP40, HSP70, HSP90, ubiquitin proteins and one copy each of cartilage associated protein (CRTAP), HSPH1, HSBP1, FK506-binding protein B (FKBP), kruppel-like factor (KLF13), peptidyl-prolyl isomerase PIPB and Prolyl 4-hydroxylase beta subunit (P4HB). This suggested a cocktail of different chaperones interact with HSP47. These findings will assist in the evaluation of roles of HSP47 in human disease including different types of cancers.
COMMUNICATION | doi:10.20944/preprints202311.0216.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: CD44; CD44 variant 4; monoclonal antibody; epitope; enzyme-linked immunosorbent assay
Online: 3 November 2023 (06:38:38 CET)
CD44 is a type I transmembrane glycoprotein, and possesses various isoforms which are largely classified into CD44 standard and CD44 variant (CD44v) isoforms. Some variant-encoded regions play critical roles in tumor progression. However, the function of CD44 variant 4 (CD44v4)-encoded region has not been fully understood. Using peptide immunization, we developed an anti-CD44v4 mAb, C44Mab-108, which is useful for flow cytometry, western blotting, and immunohistochemistry. In this study, we determined the critical epitope of C44Mab-108 by enzyme-linked immunosorbent assay (ELISA). We used the alanine (or glycine)-substituted peptides of the CD44v4-encoded region (amino acids 271-290 of human CD44v3-10), and found that C44Mab-108 did not recognize the alanine-substituted peptides of D280A and W281A. Furthermore, these peptides could not inhibit the recognition of C44Mab-108 in flow cytometry and immunohistochemistry. The results indicate that the critical binding epitope of C44Mab-108 includes Asp280 and Trp281 of CD44v3-10.
REVIEW | doi:10.20944/preprints202307.1900.v1
Subject: Biology And Life Sciences, Animal Science, Veterinary Science And Zoology Keywords: DNA vaccine; delivery system, variant surface glycoprotein; nanoparticle; antigenic variation; endemic
Online: 28 July 2023 (02:27:14 CEST)
Trypanosomes are the extracellular protozoan parasites that cause human African trypanosomiasis disease in humans and nagana disease in animals. The disease is endemic in African countries where tsetse flies which act as a vector for the transmission of disease are present. The animals infected by these parasites become useless or workless and disease can become fatal if not treated. Old drugs used for treatment have many side effects and some are responsible for death in 5% of the patients. The parasite possesses a major surface protein known as variant surface glycoprotein. The immune system of the host develops antibodies against this antigen but due to antigenic variation, parasites evade the immune response. Currently, no vaccine is available that provides complete protection. Only partial protection using certain antigens in murine models was investigated. The tools of molecular biology and immunology have enabled a novel approach to the development of vaccines against trypanosomes. Immunization is the sole method for the control of disease because the eradication of the vector from endemic areas is an impossible task. Genetic vaccines can carry multiple genes encoding different antigens of the same parasite or different parasites. DNA immunization induces the activation of both cellular immune response and humoral immune response along with the generation of memory. This review highlights the importance of DNA vaccines and advances in the development of DNA vaccines against T. brucei.
ARTICLE | doi:10.20944/preprints202306.0059.v1
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: COVID-19; SARS-CoV-2; remdesivir; nirmatrelvir; variant; synergy; antiviral; immunosuppressed
Online: 1 June 2023 (08:00:38 CEST)
Introduction: This study aims to investigate the activity of the remdesivir-nirmatrelvir combination against Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) and to report a case of Coronavirus Disease 2019 (COVID-19) cured with this combination. Methods: A Vero E6 cell-based infection assay was used to investigate the in vitro activity of the remdesivir-nirmatrelvir combination. The SARS-CoV-2 strains tested were 20A.EU1, BA.1 and BA.5. After incubation, a viability assay was performed. The supernatants were collected and used for viral titration. The Highest Single Agent (HSA) reference model was calculated. An HSA score >10 is considered synergic. Results: Remdesivir and nirmatrelvir showed synergistic activity at 48 and 72 h with an HSA score of 52.8 and 28.6, respectively (p<0.0001). This data has been confirmed performing supernatant titration and against the omicron variants: the combination reduced the viral titer better than the more active compound alone. An immunocompromised patient with prolonged and critical COVID-19 was successfully treated with remdesivir, nirmatrelvir/ritonavir, tixagevimab/cilgavimab, and dexamethasone, with an excellent clinical-radiological response. However, she required further off-label prolonged therapy with nirmatrelvir/ritonavir up to the negativization. Conclusion: Remdesivir-nirmatrelvir combination has synergic activity in vitro. This combination may have a role in immunosuppressed patients with severe COVID-19 and prolonged viral shedding.
ARTICLE | doi:10.20944/preprints202305.1361.v1
Subject: Biology And Life Sciences, Virology Keywords: COVID-19; SARS-CoV-2; Omicron Variant of Concern; Mutations; Lineages
Online: 19 May 2023 (03:00:56 CEST)
The emergence of the SARS-CoV-2 Variant of Concern (VOC) Omicron has been characterized by an explosive number of cases in almost every part of the world. The dissemination of different sub-lineages and recombinant genomes also led to several posterior waves in many countries. The circulation of this VOC and its major sub-lineages (BA.1 to BA.5) was monitored in community cases and in international travelers returning to Venezuela, by a rapid partial sequencing method. The specific sub-lineage assignment was performed by complete genome sequencing. Epidemic waves of SARS-CoV-2 cases were observed among international travelers during 2022, a situation not seen before December 2021. The succession of the Omicron VOC sub-lineages BA.1 to BA.5 occurred sequentially, except for BA.3, which was almost not detected. However, the sub-lineages generally circulated two months earlier in international travelers than in community cases. The diversity of Omicron sub-lineages found in international travelers was related to the one found in the USA, consistent with the most frequent destination of international travel from Venezuela this year. These differences are compatible with the delay observed in Latin American countries in the circulation of the different lineages of the Omicron VOC.
REVIEW | doi:10.20944/preprints202305.0103.v1
Subject: Biology And Life Sciences, Virology Keywords: SARS-CoV-2; antiviral agents; Omicron variant; mutant; vaccines; clinical studies
Online: 3 May 2023 (06:04:36 CEST)
Since the onset of coronavirus disease 2019 (COVID-19) pandemic, the virus kept developing and mutating into different variants over time that also gained increased transmissibility and spread in populations at a higher pace, culminating in successive waves of COVID-19 cases. The scientific community has developed vaccines and antiviral agents against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) disease. Realizing that growing SARS-CoV-2 variations significantly impact the efficacy of antiviral therapies and vaccines, we have summarized the appearance and attributes of SARS-CoV-2 variants for future perspectives in drug design, bringing up-to-date hints for developing therapeutic agents targeting the variants. The Omicron variant is among the most mutated form; its strong transmissibility and immune resistance capacity have prompted international worry. Most mutation sites currently being studied are located in the BCOV_S1_CTD of the S protein. Despite this, several hurdles remain, such as developing vaccination and pharmacological treatment efficacies for emerging mutants of SARS-CoV-2 strains. In this review, we present an updated viewpoint on the current issues faced by the emergence of various SARS-CoV-2 variants. Furthermore, we discuss the clinical studies conducted to assist the development and dissemination of vaccines, small molecule therapeutics, and therapeutic antibodies having broad-spectrum action against SARS-CoV-2 strains.
ARTICLE | doi:10.20944/preprints202304.0280.v1
Subject: Medicine And Pharmacology, Urology And Nephrology Keywords: Androgen receptor; androgen receptor splice variant 7; prostate cancer; localization; NanoBiT
Online: 13 April 2023 (02:54:24 CEST)
The androgen/androgen receptor (AR)-signaling axis plays a central role in the development and growth of prostate cancer (PCa) cells. Upon androgen-binding the AR dimerizes with another AR, translocates into the nucleus where the AR-dimer activates/inactivates androgen-dependent genes. In consequence treatments for locally advanced or metastatic PCa are commonly based on androgen deprivation therapies (ADT). Unfortunately, the clinical benefits of ADT are only transitory and most tumors develop mechanisms allowing the AR to bypass its need for physiological levels of circulating androgens. In the clinic failure of ADT is often characterized by the synthesis of a C-terminally truncated, constitutively active AR splice variant, termed AR-V7. In contrast to AR, the constitutively active AR-V7 does no longer need androgenic stimuli for nuclear entry and/or dimerization. The goal of the present study was to mechanistically decipher the interaction between full-length AR (AR-FL) and AR-V7 in AR-null HEK-293 cells using the NanoLuc Binary Technology (NanoBiT) structural complementation assay under androgen stimulation and deprivation conditions. Our data point toward a hypothesis that AR-FL/AR-FL homodimers form in the cytoplasm, whereas AR-V7/AR-V7 localize in the nucleus. However, after 15 min of androgen stimulation, all AR-FL/AR-FL, AR-FL/AR-V7 and AR-V7/AR-V7 dimers localized in the nucleus. In this way, we can show an androgen-regulated interaction between AR-FL and AR-V7 at forming heterodimers that localize in the nucleus, whereas AR-V7/AR-V7 dimers were found to localize in the absence of androgens in the nucleus. Treatment with enzalutamide diminished the luminescence of AR-FL homodimers and AR-FL/AR-V7 heterodimers but not AR-V7/AR-V7 homodimers.
ARTICLE | doi:10.20944/preprints202101.0330.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: germline; rare variant; cancer; lymphoid; B-cell; lymphomal; CLL; driver; prognosis
Online: 18 January 2021 (12:14:32 CET)
Growing evidence has revealed the implication of germline variation in cancer predisposition and prognostication. Here, we describe an analysis of putatively disruptive rare variants across the genomes of 726 patients with B-cell lymphoid neoplasms. We discovered a significant enrichment of 26 genes in germline protein truncating variants (PTVs), affecting cell signaling (MET, JAK2, ANGPT2), energy metabolism (ACO1) and nucleic acid metabolism and repair pathways (NT5E, DCK). Interestingly, some of these variants were restricted to either chronic lymphocytic leukemia (CLL) (i.e., ANGPT2 and AKR1C3) or B-cell lymphoma cases (PNMT, TPT1 and IGHMBP2). Additionally, we detected 1,675 likely disrupting variants in genes associated with cancer, of which 44.75% were novel events and 7.88% were PTVs. Among these, the most frequently affected genes were ATM, BIRC6, CLTCL1A and TSC2. Homozygous or compound heterozygous variants were detected in 28 cases; and coexisting somatic events were observed in 17 patients, some of which affected key lymphoma drivers such as ATM, KMT2D and MYC. Finally, we observed that variants in the helicase gene WRN were independently associated with shorter survival in CLL. Our study results support an important role for rare germline variation in the pathogenesis, clinical presentation and disease outcome of B-cell lymphoid neoplasms.
ARTICLE | doi:10.20944/preprints202310.0409.v1
Subject: Medicine And Pharmacology, Clinical Medicine Keywords: Stroke; COVID-19; SARS-CoV-2 Omicron variant; modified Ranking scale; coagulopathy
Online: 9 October 2023 (09:30:50 CEST)
COVID-19 has rapidly spread worldwide since December 2019. During the pandemic, SARS-CoV-2 repeatedly mutated from Alpha to Omicron. The severity and mortality are related to age, sex, and underlying diseases (respiratory, cerebrovascular, cardiovascular, metabolic, cancer, and immune diseases). We retrospectively studied 44 hospitalized stroke patients infected with the SARS-CoV-2 Omicron variant. The mortality rate of hospitalized stroke patients with COVID-19 was 6 (13.6%). The cause of death was coagulopathy in 3 cases of ischemic stroke. High modified Rankin Scale score（mRS）of stroke patient was correlated with severity of COVID-19. The timing of vaccination is inversely correlated with COVID-19 severity. In conclusion, stroke patients with COVID-19 have high mortality rates attributed to coagulopathy. Stroke patients with high mRS scores are more likely to develop severe COVID-19. In high mRS scores, therapy for severe COVID-19 should be administered.
ARTICLE | doi:10.20944/preprints202306.0199.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: mutations; folding free energy change; binding free energy change; single nucleotide variant
Online: 2 June 2023 (11:58:21 CEST)
Development of methods and algorithms to predict the effect of mutations on protein stability, protein-protein, and protein-DNA/RNA binding is necessitated by the needs of protein engineering and for understanding the molecular mechanism of disease-causing variants. The vast majority of the leading methods are either methods with adjustable parameters or machine learning algorithms, both requiring a database of experimentally measured folding and binding free energy changes. These databases are collections of experimental data taken from scientific investigations typically aimed at probing the role of particular residue on the above-mentioned thermodynamics characteristics, i.e., the mutations are not introduced at random and do not necessarily represent mutations originating from single nucleotide variant (SNV). Thus, the reported performance of the leading algorithms assessed on these databases or other limited cases, may not be applicable for predicting the effect of SNVs seen in the human population. Indeed, we demonstrate that the SNVs and non-SNVs are not equally presented in the corresponding databases and the distribution of the free energy changes are not the same. Furthermore, the Pearson correlation coefficients (PCCs) obtained on cases involving SNVs are less impressive than for non-SNVs, indicating that caution should be used in applying them to reveal the effect of human SNVs. All methods are found to underestimate the energy changes by roughly a factor of 2.
ARTICLE | doi:10.20944/preprints202206.0255.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: coronaviruses; SARS-CoV-2; variant; Omicron; SLiMs; spike protein; motifs; covid-19
Online: 17 June 2022 (13:49:02 CEST)
Short Linear Motifs (SLiMs) are short linear sequences that can mediate protein-protein interaction. Mimicking eukaryotic SLiMs to compete with extra or intracellular binding partners or to sequester host proteins is the crucial strategy of viruses to pervert the host system. The evolved proteins in viruses facilitate minimal protein-protein interactions that significantly affect intracellular signaling networks. Unfortunately, very little information about the SARS-CoV-2 SLiMs is known, especially across the SARS-CoV-2 variants. Through ELM database-based sequence analysis of spike protein from all the major SARS-CoV-2 variants, we identified four overriding SLiMs in the SARS-CoV-2 Omicron variant including LIG_TRFH_1, LIG_REV1ctd_RIR_1, LIG_CaM_NSCaTE_8, and MOD_LATS_1. These SLiMs are highly likely to interfere with various immune functions, interact with host intracellular proteins, regulate cellular pathways, and lubricate viral infection and transmission. These cellular interactions possibly serve as potential therapeutic targets for these variants, and this approach can be further exploited to combat emerging SARS-CoV-2 variants.
ARTICLE | doi:10.20944/preprints202112.0473.v1
Subject: Medicine And Pharmacology, Pathology And Pathobiology Keywords: Fanconi anemia; Chromosome instability; FANCG; splicing; founder pathogenic variant; Mixe indigenous group.
Online: 29 December 2021 (19:28:49 CET)
Fanconi anemia (FA) is a rare genetic disorder caused by pathogenic variants (PV) in at least 22 genes, which cooperate in the FA/BRCA pathway to maintain genome stability. PV in FANCA, FANCC, and FANCG account for most cases (~90%). This study evaluated the chromosomal, molecular, and phenotypic findings of a novel founder FANCG PV, identified in three patients with FA from the Mixe community of Oaxaca, Mexico. All patients presented chromosomal instability and a homozygous PV, FANCG: c.511-3_511-2delCA, identified by next-generation sequencing analysis. Bioinformatics predictions suggest that this deletion disrupts a splice acceptor site promoting the exon 5 skipping. Analysis of Cytoscan 750K arrays for haplotyping and global ancestry supported the Mexican origin and founder effect of the variant, reaffirming the high frequency of founder PV in FANCG. The degree of bone marrow failure and physical findings (described through the acronyms VACTERL-H and PHENOS) were used to depict the phenotype of the patients. Despite having a similar frequency of chromosomal aberrations and genetic constitution, the phenotype showed a wide spectrum of severity. The identification of a founder PV could help for a systematic and accurate genetic screening of patients with FA suspicion in this population.
COMMUNICATION | doi:10.20944/preprints201909.0346.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: neurogenetics; dementia; next-generation sequencing; in silico analysis; genetic variant; phenotypic variability
Online: 30 September 2019 (11:05:36 CEST)
Background: Advancements in the next-generation sequencing (NGS) techniques have allowed for efficient genetic variant detection at reduced costs. Methods: We describe an ad hoc NGS-based custom designed resequencing gene panel to identify genetic variants in 8 patients with dementing disorders. Results: We found variants of TREM2 and APP genes in three patients; these have been previously identified as pathogenic or likely pathogenic and, therefore, considered as “Disease Causing”. In the remaining subjects, the pathogenicity was evaluated on the in silico analysis, according to the guidelines of the American College of Medical Genetics. In one patient, the p.R205W variant was causative of the disease, thus considered as “Possibly Disease Causing”. The variants found from the other four subjects in the CSF1R, SERPINI1, GRN, and APP genes revealed discordant in silico results and, therefore, it was not possible to assign a definitive pathogenicity. Conclusions: Notwithstanding the limitations of a customized panel, we detected some rare genetic variants with a probable disease association. The future application of NGS techniques and the further replication of these experimental data will replace the so-called “gene by gene” approach with a “panel of genes” strategy, that offers promising perspectives in the diagnosis and management of neurodegenerative disorders.
REVIEW | doi:10.20944/preprints202308.0822.v1
Subject: Biology And Life Sciences, Neuroscience And Neurology Keywords: behavioral variant of frontotemporal dementia; correlates; functional impairment; predictive factors; PRISMA; systematic review
Online: 10 August 2023 (05:47:33 CEST)
The behavioral variant of frontotemporal dementia (bvFTD) has a devastating effect on multiple domains of daily living. The purpose of this PRISMA-compliant systematic review is to summarize the most important factors associated with functional impairment in this clinical group by critically analyzing the existing literature spanning the period from 2000 to 2023. To be included in the review, the study had to investigate any kind of correlates of functional status in bvFTD patients, using a previously validated instrument of functional assessment. Out of 40 articles assessed for eligibility, 18 met the inclusion criteria. The anatomical pattern of cerebral atrophy at baseline appeared to be the strongest predictor of the rate of functional decline over time, with the frontal-dominant anatomical subtype being associated with a faster rate of functional impairment. Additionally, executive dysfunction as well as apathy appeared to contribute significantly to functional disability in bvFTD patients. A comparative examination of bvFTD in relation to other clinical subtypes of FTD and other types of dementia in general suggests that it’s the predominant atrophy of the frontal lobes along with the subsequent unique combination of cognitive and neuropsychiatric manifestations that account for the pronounced functional limitations observed in these individuals, even from the early stages of the disease.
ARTICLE | doi:10.20944/preprints202209.0318.v1
Subject: Biology And Life Sciences, Virology Keywords: BA.2.75 variant; Gibbs energy of binding; binding rate; infectivity; SARS-CoV-2
Online: 21 September 2022 (09:44:34 CEST)
Omicron BA.2.75 may become the next globally dominant strain of COVID-19 in 2022. BA.2.75 sub-variant has acquired more mutations (9) in spike protein and other genes of SARS-CoV-2 than any other variant. Thus, its chemical composition and thermodynamic properties have changed comparing to earlier variants. In this paper Gibbs energy of binding and antigen-receptor binding rate is reported for the BA.2.75 variant. Gibbs energy of binding (driving force of binding) of Omicron BA.2.75 variant is more negative than that of the competing variants BA.2 and BA.5.
ARTICLE | doi:10.20944/preprints202201.0466.v1
Subject: Medicine And Pharmacology, Ophthalmology Keywords: FOXC1; in vitro studies; novel variant; ophthalmic genetics; intrafamilial variability; anterior segment dysgenesis
Online: 31 January 2022 (13:36:18 CET)
Anterior segment dysgenesis (ASD) encompasses a wide spectrum of developmental abnormalities of the anterior ocular segment, including congenital cataract, iris hypoplasia, aniridia, iridocorneal synechiae, as well as Peters, Axenfeld, and Rieger anomalies. Here, we report a large five-generation Caucasian family exhibiting atypical syndromic ASD segregating with a novel truncating variant of FOXC1. The family history is consistent with highly variable autosomal dominant symptoms including isolated glaucoma, iris hypoplasia, aniridia, cataract, hypothyroidism, congenital heart anomalies, and cystic kidney disease. Whole exome sequencing revealed a novel variant [c.313_314insA; p.(Tyr105*)] in FOXC1 that disrupts the alpha-helical region of the DNA-binding forkhead box domain. In vitro studies using a heterologous cell system revealed aberrant cytoplasmic localization of FOXC1 harboring the Tyr105* variant, likely precluding downstream transcription function. Meta-analysis of the literature highlighted the intrafamilial variability related to FOXC1 truncating alleles. This study highlights the clinical variability in ASD and signifies the importance of combining both clinical and molecular analysis approaches to establish a complete diagnosis.
ARTICLE | doi:10.20944/preprints202309.1964.v1
Subject: Biology And Life Sciences, Virology Keywords: Biothermodynamics; Virus-host interaction; COVID-19; Pandemic; Variant under monitoring; Pathogenicity; Pathogen; August 2023
Online: 28 September 2023 (11:23:34 CEST)
Similarly to a phoenix, SARS-CoV-2 has appeared periodically in waves. The new variants that appeared through mutations have during the 4 years of the pandemic suppressed earlier variants, causing new waves of the pandemic. The Omicron BA.2.86 Pirola variant is the latest in the sequence of SARS-CoV-2 variants, which appeared in 2023. The BA.2.86 variant has started to spread rapidly and we are witnesses of a new epidemic wave. In this short period, an increased infectivity was noticed, which results in rapid spreading and decreased pathogenicity, which results in a lower number of severe cases. However, in the public there is a fear of further development of the epidemic. This analysis was made with the goal to assess the risks in the period of late 2023. Mutations that were developed by the BA.2.86 variant have led to a change in empirical formula and thermodynamic properties. It seems that there is no ground for fear of an extensive spreading of severe forms, but there are reasons for caution and monitoring of the spreading of the epidemic and potential appearance of new mutations.
ARTICLE | doi:10.20944/preprints202307.0876.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: SARS-CoV-2; P.1 variant; B.1 strain; cytokines; COVID-19; macrophages; neutrophils
Online: 13 July 2023 (04:34:23 CEST)
Since the first description of SARS-CoV-2 in China in 2019, thousands of variants have emerged worldwide. For some of them, the constellation of mutations caused changes in virus biology, pathogenicity, infectivityity and transmissibility resulting in dissemination throughout the world. Gamma variant (P.1) differs from SARS-CoV-2 Wuhan strain (B.1) by 12 amino acids in the Spike (S) protein, and presented mutations related to greater affinity for the receptor angioten-sin-converting enzyme 2 (ACE-2) and/or immune escape. The Gamma variant and subvariants were responsible for the second wave of COVID-19 in the Brazilian city of Manaus, characterized by high mortality and rapid transmission. The ability of variants to induce cytokine production may be closely related to their pathogenicity. Herein we observed that there was no significant difference in the quantity of cytokines among macrophages or neutrophils infected with P.1 and B.1 strains. Also, no significant difference was observed in the absolute number of macrophages and neutrophils infected with these variants. Furthermore, no evidence of SARS-CoV-2 replication was observed in macrophages when infected by the two analyzed variants. Our findings suggest that the difference in the epidemiological outcome observed during the P.1 variant spread when compared to B.1, it is not explained by differences in the quantity of cytokines and absolute number of macrophages or neutrophils. Through bioinformatics analysis of the S protein, we observed that the physicochemical differences between the variants and subvariants of P.1, probably refer to the degree of infectivity, due to the impact caused in the recognition of antibodies and receptor af-finity
ARTICLE | doi:10.20944/preprints202206.0272.v1
Subject: Medicine And Pharmacology, Pulmonary And Respiratory Medicine Keywords: COVID-19; SARS-CoV-2; molnupiravir; nirmatrelvir; GC376; PF-07321332; variant; synergy; an-tiviral
Online: 20 June 2022 (10:57:26 CEST)
Introduction: The development of effective vaccines has partially mitigated the trend of the SARS-CoV-2 pandemic, however, the need for orally available antiviral drugs persists. This study aims to investigate the activity of molnupiravir in combination with nirmatrelvir or GC376 on SARS-CoV-2 to verify the synergistic effect. Methods: The SARS-CoV-2 strains 20A.EU, BA.1 and BA.2 were used to infect Vero E6 in presence of antiviral compounds alone or in combinations using 5 two-fold serial dilution of compound concentrations ≤EC90. After 48 and 72 h post-infection, viability was performed using MTT reduction assay. Supernatants were collected for plaque-assay titration. All experiments were conducted three times and in triplicate. The synergistic score was calculated using Synergy Finder version 2. Results: All compounds reached micromolar EC90. Molnupiravir and GC376 showed a synergistic activity at 48 h with an HSA score of 19.33 (p<0.0001) and an additive activity at 72 h with an HSA score of 8.61 (p<0.0001). Molnupiravir and nirmatrelvir showed a synergistic activity both at 48 h and 72 h with an HSA score of 14.2 (p=0.01) and 13.08 (p<0.0001), respectively. Conclusion: Molnupiravir associated with one of the two protease-inhibitors nirmatrelvir and GC376 showed good additive-synergic activity in vitro.
CASE REPORT | doi:10.20944/preprints202204.0285.v1
Subject: Medicine And Pharmacology, Pediatrics, Perinatology And Child Health Keywords: GLDN variant; gliomedin; juvenile progressive respiratory insufficiency; diaphragmatic hypomotility; scoliosis; arthrogryposis; LCCS11; axonopathy; FADS
Online: 29 April 2022 (03:36:40 CEST)
Lethal congenital contracture syndrome 11 (LCCS11) is a form of arthrogryposis multiplex congenita (AMC) which is associated with mutations in the gliomedin gene (GLDN) and has been known to be severely life-shortening, mainly due to respiratory insufficiency. Patients with this condition have been predominantly treated by pediatricians as they usually do not survive beyond childhood. In this case report we present a young adult who developed severe progressive respiratory insufficiency as a teenager due to diaphragmatic hypomotility and was diagnosed with LCCS11 following the finding of compound heterozygous pathogenic variants in GLDN. This case demonstrates the importance of screening for neuromuscular diseases in well-child visits and follow-ups of patients at risk for gross and fine motor function developmental delay. It also underscores the significance of including LCCS11 and other axonopathies in the differential diagnosis of juvenile onset of respiratory insufficiency, highlights that patients with this condition may present to adult practitioners and questions whether the nomenclature of this condition with various phenotypes should be reconsidered due to the stigmatizing term ‘lethal’.
COMMUNICATION | doi:10.20944/preprints202203.0185.v1
Subject: Medicine And Pharmacology, Pulmonary And Respiratory Medicine Keywords: SARS-CoV-2; COVID-19; variant; sublineage; transmission; immunity; infection; vaccination; non-pharmaceutical interventions
Online: 14 March 2022 (11:19:04 CET)
The scientific, private and industrial sectors use a wide variety of technological platforms available to achieve protection against SARS-CoV-2, including vaccines. However, the virus evolves continually into new highly virulent variants, which might overcome the protection provided by vaccines and may re-expose the population to infections. Mass vaccinations should be continued in combination with more or less obligation mandatory non-pharmaceutical interventions. Therefore, the key questions to be answered are: (i) How to identify the primary and secondary infections of SARS-CoV-2? (ii) Why are neutralizing antibodies not long-lasting in both the cases of natural infections and post-vaccinations? (iii) Which are the factors responsible for this decay in neutralizing antibodies? (iv) What strategy could be adapted to develop long-term herd immunity? (v) Is the Spike the only vaccine candidate or a vaccine cocktail is better?
CASE REPORT | doi:10.20944/preprints202202.0251.v1
Subject: Medicine And Pharmacology, Cardiac And Cardiovascular Systems Keywords: Infective endocarditis; Granulicatela adiacens; Nutritionally variant streptococci (NVS); Next-generation sequencing (NGS); Aortic valve
Online: 21 February 2022 (10:45:14 CET)
In this report, we describe the course and successful treatment of complicated infective endocarditis (IE). A patient presented with a high-grade irregular fever with chills lasting at least 2 months, dyspnoea, chest pain, fatigue, weight loss, and night sweats during the previous 3 months. Above cardiac congenital disorders, he was found to have Granulicatella adiacens infective aortic valve endocarditis, presumably transmitted from the oral cavity niche. Validated metagenomic 16S rDNA next generation sequencing was used to perform taxonomic identification, allowing for specific adequate antibiotic therapy instead of empiric therapy. This paper highlights the critical role of rapid taxonomic identification of nutritionally variant streptococci crucial and the benefit of proper IE treatment to avoid relapses or fatal complications.
REVIEW | doi:10.20944/preprints202202.0224.v1
Subject: Biology And Life Sciences, Virology Keywords: COVID-19; pandemic; SARS-CoV-2; Omicron variant; emerging disease; global health; virus; genome; mutation
Online: 18 February 2022 (05:23:18 CET)
The 2019 Coronavirus Disease (COVID-19) is a major cause of morbidity and mortality worldwide. Since late November 2021, Omicron variant has emerged as the primary cause of COVID-19 and caused a huge increase in the reported incidence around the world. To date, 32-34 spike mutations have been reported, 15 of which were located in the receptor-binding domain that interacts with the cell surface of the host cells, while the rest were located in the N-terminal domain and around the Furin cleavage site. At present, both the genomic and clinical profiles of this novel variant are being investigated. Here, we aim to discuss the recent reports on the transmissibility and severity of Omicron variant from both the genetic and clinical perspectives. Afterward, we also take the chance to deliver our personal view on the topic.
ARTICLE | doi:10.20944/preprints201911.0271.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: breast cancer risk; GWAS; candidate causal variant; chromatin conformation capture; reporter gene activity; enhancer; promoter
Online: 24 November 2019 (05:12:07 CET)
Genome-wide association studies have revealed a locus at 8p12 that is associated with breast cancer risk. Fine-mapping of this locus identified 16 candidate causal variants (CCVs). However, as these variants are intergenic, their function is unclear. To map chromatin looping from this risk locus to a previously identified candidate target gene, DUSP4, we performed chromatin conformation capture analyses in normal and tumoral breast cell lines. We identified putative regulatory elements, containing CCVs, that loop to the DUSP4 promoter region. Using reporter gene assays, we found that the risk allele of CCV rs7461885 reduced the activity of a DUSP4 enhancer element, consistent with the function of DUSP4 as a tumor suppressor gene. Furthermore, the risk allele of CCV rs12155535, located in another DUSP4 enhancer element, was negatively correlated with looping of this element to the DUSP4 promoter region, suggesting that this allele would be associated with reduced expression. These findings provide the first evidence that CCV risk alleles downregulate DUSP4 expression, suggesting that this gene is a regulatory target of the 8p12 breast cancer risk locus.
ARTICLE | doi:10.20944/preprints202212.0122.v1
Subject: Biology And Life Sciences, Virology Keywords: COVID-19; Elemental composition; Gibbs energy of biosynthesis; Gibbs energy of binding; Variant of concern (VOC)
Online: 7 December 2022 (09:19:10 CET)
RNA viruses exhibit a great tendency to mutate. Starting from 2019, the wild type that was labeled Hu-1 has during the last 3 years evolved to produce several dozen new variants, as a consequence of mutations. Mutations cause changes in empirical formulas of new virus strains, which lead to change in thermodynamic properties of biosynthesis and binding. These changes cause changes in the rate of reactions of binding of virus antigen to the host cell receptor and the rate of virus multiplication in the host cell. Changes in thermodynamic and kinetic parameters lead to changes in biological parameters of infectivity and pathogenicity. SARS-CoV-2 has starting from in 2019, until today, evolved towards increase in infectivity and maintaining constant pathogenicity, or for some variants a slight decrease in pathogenicity. In the case of Omicron BQ.1, BQ.1.1, XBB and XBB.1 variants pathogenicity is identical as in the Omicron BA.2.75 variant. On the other hand, infectivity of the Omicron BQ.1, BQ.1.1, XBB and XBB.1 variants is greater than those of previous variants. This will most likely result in the phenomenon of asymmetric coinfection, that is circulation of several variants in the population, some being dominant.
Subject: Biology And Life Sciences, Virology Keywords: foot-and-mouth disease virus; vaccine efficacy; serotype O/ME-SA/Ind2001 variant; heterologous challenge; cattle
Online: 23 August 2021 (14:17:25 CEST)
Vaccination is one of the best approaches to control and eradicate foot-and-mouth disease (FMD). To achieve this goal, vaccines with inactivated FMD virus antigen in suitable adjuvants are being used in addition with other control measures. However, only a limited number of vaccine strains are commercially available which often have a restricted spectrum of activity against the different FMD virus strains in circulation. As a result, when new strains emerge, it is important to measure the efficacy of the current vaccine strains against these new variants. This is important for countries where FMD is endemic but also for countries that hold an FMD vaccine bank to be prepared for emergency vaccination. The emergence and spread of the O/ME-SA/Ind-2001 lineage of viruses posed a serious threat to countries which had OIE-endorsed FMD control plans and had not reported FMD for many years. In vitro vaccine matching results showed a poor match (r1-value <0.3) to the more widely used vaccine strain O Manisa and less protection in a challenge test. This paper describes the use of O3039 vaccine strain as an alternative either alone or in combination with the O Manisa vaccine strain with virulent challenge by a O/ME-SA/Ind-2001d sub-lineage virus from Algeria (O/ALG/3/2014). The experiment included challenge at 7 days post vaccination (to study protection and emergency use) and 21 days post vaccination (as would be done in standard potency studies). The results indicated that the O3039 vaccine strain alone as well as the combination with O Manisa is effective against this strain of the O/ME-SA/Ind/2001d lineage offering protection from clinical disease even after 7 days post vaccination and with reduction in viraemia and virus excretion.
BRIEF REPORT | doi:10.20944/preprints202004.0024.v1
Subject: Biology And Life Sciences, Virology Keywords: COVID-19; SARS-nCoV-2; vaccine; antibody; immune escape; variant; spike protein; genomic drift; convalescent plasma
Online: 3 April 2020 (04:24:52 CEST)
New coronavirus (SARS-CoV-2) treatments and vaccines are under development to combat the COVID-19 disease. Several approaches are being used by scientists for investigation including 1) various small molecule approaches targeting RNA polymerase, 3C-like protease, and RNA endonuclease and 2) exploration of antibodies obtained from convalescent plasma from patients who have recovered from COVID-19. The coronavirus genome is highly prone to mutations that lead to genetic drift and escape from immune recognition; thus, it is imperative that sub-strains with different mutations are also accounted for during vaccine development. As the disease has grown to become a pandemic, new B-cell and T-cell epitopes predicted from SARS coronavirus have been reported. Using the epitope information along with variants of the virus, we have found several variants which might cause drifts. Among such variants, 23403A>G variant (p.D614G) in spike protein B-cell epitope is observed frequently in European countries such as the Netherlands, Switzerland and France.
CONCEPT PAPER | doi:10.20944/preprints202207.0408.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Lynch syndrome variant heterozygotes; colorectal and non-colorectal cancers; frameshift muta-tions; neoantigens; immune responses; immunogenomic biomarkers
Online: 26 July 2022 (10:58:51 CEST)
Lynch syndrome (LS) is an inherited disorder in which affected individuals have a significantly higher-than-average risk of developing colorectal and non-colorectal cancers, often before the age of 50 years. In LS, mutations in DNA repair genes lead to a dysfunctional post-replication repair system. As a result, the unrepaired errors in coding regions of the genome produce novel proteins, called neoantigens. Neoantigens are recognised by the immune system as foreign and trigger an immune response. Due to the invasive nature of cancer screening tests, universal cancer screening guidelines unique for LS (including colonoscopy) are poorly adhered to by LS variant heterozygotes (LSVH). Currently, it is unclear whether immunogenomic components produced as a result of neoantigen formation can be used as novel biomarkers in LS. We hypothesize that: (i) LSVH produce measurable and dynamic immunogenomic components in blood, and (ii) these quantifiable immunogenomic components correlate with cancer onset and stage. Here, we discuss feasibility and propose the potential to: (a) identify personalised novel immunogenomic biomarkers, (b) reduce invasive cancer screening tests and thus increase non-invasive cancer surveillance, (c) improve compliance and adherence to recommended cancer screening guidelines in LSVH.
ARTICLE | doi:10.20944/preprints201811.0183.v2
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: sequencing technologies; NGS; genome research; genome assembly; variant calling; RNA-Seq; transcriptome assembly; bioinformatics; molecular biology; education
Online: 13 November 2018 (10:22:06 CET)
Combined awareness about the power and limitations of bioinformatics and molecular biology enables advanced research based on high-throughput data. Despite an increasing demand for scientists with a combined background in both fields, the education in dry lab and wet lab is often separated. This work describes an example of integrated education with focus on genomics and transcriptomics. Participants learn computational and molecular biology methods in the same practical course. Peer-review is applied as a teaching method to foster cooperative learning of students with heterogeneous backgrounds. Evaluation results indicate acceptance and appreciation of this approach.
ARTICLE | doi:10.20944/preprints202309.2171.v1
Subject: Medicine And Pharmacology, Epidemiology And Infectious Diseases Keywords: Tixagevimab/Cilgavimab, kidney transplantation, SARS-CoV-2, COVID-19, Kraken variant, XBB.1.5, pre-exposure prophylaxis, immunosuppressed patients
Online: 2 October 2023 (11:22:50 CEST)
Pre-exposure prophylaxis for COVID-19 with Tixagevimab/Cilgavimab in immunocompromised patients has reduced the risk of breakthrough infection, disease, hospitalization, and COVID-19 related mortality. However, the advent of the Kraken variant (XBB.1.5) has limited the use of this monoclonal antibody, based on poor efficacy in in vitro studies The objective of the study was to evaluate the risk of breakthrough infection, symptomatic disease, hospitalization, intensive care admission and COVID-19 related death in kidney transplanted recipients receiving pre-exposure prophylaxis with Tixagevimab/Cilgavimab for COVID-19 in the era of the Kraken variant (XBB.1.5). In a prospective, observational study, we enrolled kidney transplant patients undergoing pre-exposure prophylaxis for COVID-19 with Tixagevimab/Cilgavimab at the Division of Infectious Diseases of Federico II University of Naples between February 2023 and August 2023. Each patient subsequently underwent a six-month follow-up with symptom monitoring and surveillance nasopharyngeal swab for SARS-CoV-2 RNA detection every 30 days, regardless of symptoms. Thirty-four kidney transplant patients were enrolled and in the follow-up period, only one tested positive for the nasopharyngeal swab for SARS-CoV-2 research with asymptomatic infection and virological recovery on the eighth day from the diagnosis of infection. Therefore, no patient developed disease and no patient needed hospitalization and no death occurred. No adverse drug reaction to Tixagevimab/Cilgavimab occurred. Our data, although derived from a limited and uncontrolled sample, show the potential of Tixagevimab/Cilgavimab as a valid and viable therapeutic strategy in pre-exposure prophylaxis for immunocompromised patients. These findings highlight the importance of conducting clinical studies on this topic.
ARTICLE | doi:10.20944/preprints202207.0286.v1
Subject: Biology And Life Sciences, Immunology And Microbiology Keywords: SARS-CoV-2; Omicron variant of concern; homologous boosting; heterologous boosting; Coro-naVac; BNT162b2; healthcare worker; return-to-work
Online: 19 July 2022 (10:15:39 CEST)
Immune escape is observed with SARS-CoV-2 Omicron (Pango lineage B.1.1.529), the predominant circulating strain worldwide. Booster dose was shown to restore immunity against Omicron infection, however, real world data comparing mRNA (BNT162b2; Comirnaty) and inactivated vaccine (CoronaVac; Sinovac) homologous and heterologous boosting is lacking. A retrospective study was performed to compare the rate and outcome of COVID-19 in healthcare workers (HCWs) with various vaccination regime during a territory-wide Omicron outbreak in Hong Kong. During the study period 1 Feb – 31 Mar 2022, 3167 HCWs were recruited, 871 HCWs reported 746 and 183 episodes of significant household and non-household close contact. 737 HCWs acquired COVID-19 which were all clinically mild. Time dependent Cox regression showed that, comparing with 2-dose vaccination, 3-dose vaccination reduced infection risk by 31.7% and 89.3% in household contact and non-household close contact respectively. Using 2-dose BNT162b2 as reference, 2-dose CoronaVac recipient had significantly higher risk of being infected (HR 1.69 P<0.0001). Three-dose BNT162b2 (HR 0.4778 P<0.0001) and 2-dose CoronaVac + BNT162b2 booster (HR 0.4862 P=0.0157) were associated with lower risk of infection. Three-dose CoronaVac and 2-dose BNT162b2 + CoronaVac booster were not significantly different from 2-dose BNT162b2. The mean time to achieve negative RT-PCR or E gene cycle threshold 31 or above was not affected by age, number of vaccine dose taken, vaccine type and timing of the last dose. In summary, we have demonstrated lower risk of breakthrough SARS-CoV-2 infection in HCWs given BNT162b2 as booster after 2 doses of BNT162b2 or CoronaVac.
ARTICLE | doi:10.20944/preprints202206.0192.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: eCDC; WHO; Sanger sequencing; Omicron variant; minor subvariants; BA.4/BA.5; BA.2; mul-ti-allelic; SNPs; recombinant
Online: 14 June 2022 (04:30:41 CEST)
Large population passages of the SARS-CoV-2 in the past two and a half years have allowed the circulating virus to accumulate an increasing number of mutations in its genome. The most recently emerging Omicron subvariants have the highest number of mutations in the Spike (S) protein gene and these mutations mainly occur in the receptor-binding domain (RBD) and the N-terminal domain (NTD) of the S gene. The eCDC and the WHO recommend partial Sanger sequencing of the SARS-CoV-2 S gene RBD and NTD on the PCR-positive samples in diagnostic laboratories as a practical means of determining the variants of concern to monitor a possible increased transmissibility, increased virulence, or reduced effectiveness of vaccines against them. The author’s diagnostic laboratory has implemented the eCDC/WHO recommendation by sequencing a 398-base segment of the N gene for the definitive detection of SARS-CoV-2 in clinical samples, and sequencing a 445-base segment of the RBD and a 490-509-base segment of the NTD for variant determination. This paper presents 5 selective cases to illustrate the challenges of using Sanger sequencing to diagnose Omicron subvariant when the samples harbor a high level of co-existing minor subvariant sequences with multi-allelic single nucleotide polymorphisms (SNPs) or possible recombinant Omicron subvariants containing a BA.1 NTD and a BA.2 RBD, which can only be detected by using specially designed PCR primers. The current large-scale surveillance programs using next-generation sequencing (NGS) do not face similar problems because NGS focuses on deriving consensus sequence.
REVIEW | doi:10.20944/preprints201902.0090.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: BRCA1; variants of uncertain clinical significance; VUS; germline variants; hereditary breast and ovarian cancer; breast cancer; genetic testing, ovarian cancer; variant classification; clinical annotation
Online: 11 February 2019 (16:12:03 CET)
Genetic testing allows for identification of germline DNA variations which are associated with a significant increase in risk of developing breast and ovarian cancer. Detection of a BRCA1 or BRCA2 pathogenic variant triggers several clinical management actions, which may include increased surveillance and prophylactic surgery for healthy carriers or treatment with PARP inhibitor therapy for carriers diagnosed with cancer. Thus, standardized validated criteria for annotation of BRCA1 and BRCA2 variants according to their pathogenicity are necessary to support clinical decision making and ensure improved outcomes. Upon detection, variants whose pathogenicity can be inferred by the genetic code are typically classified as pathogenic, likely pathogenic, likely benign, or benign. Variants whose impact on function cannot be directly inferred by the genetic code are labeled as Variants of Uncertain Clinical Significance (VUS) and are evaluated by multifactorial likelihood models that use personal and family history of cancer, segregation data, prediction tools, and co-occurrence with a pathogenic BRCA variant. Missense variants, coding alterations that replace a single amino acid residue with another, are a class of variants for which determination of clinical relevance is particularly challenging. Here, we discuss current issues in variant classification by following a typical life cycle of a BRCA1 missense variant through detection, annotation and information dissemination. Advances in massively parallel sequencing have led to a substantial increase in VUS findings. Although comprehensive assessment and classification of missense variants according to their pathogenicity remains the bottle neck, new developments in functional analysis, high throughput assays, data sharing, and statistical models are rapidly changing this scenario.
COMMUNICATION | doi:10.20944/preprints202106.0091.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: COVID-19; SARS-CoV-2 variant; lateral flow immunoassay; spike protein; receptor binding domain (RBD); neutralizing antibody; therapeutic antibody cocktail; epitope binning; rapid neutralization test; ACE2
Online: 2 June 2021 (16:11:29 CEST)
Identifying anti-spike antibodies that exhibit strong neutralizing activity against current dominant circulating variants and antibodies that are escaped by these variants have important implications in the development of therapeutic and diagnostic solutions as well as in improving understanding of the humoral response to SARS-CoV-2 infection. We characterized seven anti-RBD monoclonal antibodies for their binding activity, pairing capability and neutralization activity to SARS-CoV-2 and three variant RBDs (UK, SA and BR P.1) via lateral flow immunoassays. The results allowed us to group these antibodies into three distinct epitope bins. Our studies showed that two antibodies had broadly potent neutralizing activity against SARS-CoV-2 and these variant RBDs and that one antibody did not neutralize the SA and BR P.1 RBDs. The antibody escaped by the SA and BR P.1 RBDs retained binding activity to SA and BR P.1 RBDs but was unable to induce neutralization. Further, we demonstrated that the lateral flow immunoassay can be a rapid and effective tool for antibody characterization, including epitope classification and antibody neutralization kinetics. From these studies, the potential contributions of the mutations (N501Y, E484K and K417N/T) contained in these variants’ RBDs on antibody pairing capability, neutralization activity and therapeutic antibody targeting strategy are discussed.
ARTICLE | doi:10.20944/preprints202109.0415.v2
Subject: Biology And Life Sciences, Virology Keywords: Delta variant; Variants of concern; Variants of interest; SARS-CoV-2; Spike protein; Nested RT-PCR; Sanger sequencing; Amino acid mutations; ACE2 RBD; N-terminal domain (NTD)
Online: 2 November 2021 (10:40:46 CET)
As SARS-CoV-2 continues to spread among human populations, genetic changes occur and accumulate in the circulating virus. Some of these genetic changes have caused amino acid mutations, including deletions, which may have potential impact on critical SARS-CoV-2 countermeasures, including vaccines, therapeutics, and diagnostics. Considerable efforts have been made to categorize the amino acid mutations of the angiotensin-converting enzyme 2 (ACE2) receptor binding domain (RBD) of the spike (S) protein along with certain mutations in other regions within the S protein as specific variants in an attempt to study the relationship between these mutations and the biological behavior of the virus. However, the currently used whole genome sequencing surveillance technologies can test only a small fraction of the positive specimens with high viral loads and often generate uncertainties in nucleic acid sequencing that needs additional verification for precision determination of mutations. This article introduces a generic protocol to routinely sequence a 437-bp nested RT-PCR cDNA amplicon of the ACE2 RBD and a 490-bp nested RT-PCR cDNA amplicon of the N-terminal domain (NTD) of the S gene for detection of the amino acid mutations needed for accurate determination of all variants of concern and variants of interest according to the definitions published by the U.S. Centers for Disease Control and Prevention. This protocol was able to amplify both nucleic acid targets into cDNA amplicons to be used as templates for Sanger sequencing on all 16 clinical specimens that were positive for SARS-CoV-2.