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International Journal of Molecular... Jun 2024Ischemic stroke is a major cause of mortality worldwide. Proper etiological subtyping of ischemic stroke is crucial for tailoring treatment strategies. This study...
Ischemic stroke is a major cause of mortality worldwide. Proper etiological subtyping of ischemic stroke is crucial for tailoring treatment strategies. This study explored the utility of circulating microRNAs encapsulated in extracellular vesicles (EV-miRNAs) to distinguish the following ischemic stroke subtypes: large artery atherosclerosis (LAA), cardioembolic stroke (CES), and small artery occlusion (SAO). Using next-generation sequencing (NGS) and machine-learning techniques, we identified differentially expressed miRNAs (DEMs) associated with each subtype. Through patient selection and diagnostic evaluation, a cohort of 70 patients with acute ischemic stroke was classified: 24 in the LAA group, 24 in the SAO group, and 22 in the CES group. Our findings revealed distinct EV-miRNA profiles among the groups, suggesting their potential as diagnostic markers. Machine-learning models, particularly logistic regression models, exhibited a high diagnostic accuracy of 92% for subtype discrimination. The collective influence of multiple miRNAs was more crucial than that of individual miRNAs. Additionally, bioinformatics analyses have elucidated the functional implications of DEMs in stroke pathophysiology, offering insights into the underlying mechanisms. Despite limitations like sample size constraints and retrospective design, our study underscores the promise of EV-miRNAs coupled with machine learning for ischemic stroke subtype classification. Further investigations are warranted to validate the clinical utility of the identified EV-miRNA biomarkers in stroke patients.
Topics: Humans; Machine Learning; Ischemic Stroke; Male; Circulating MicroRNA; Female; Aged; Middle Aged; Exosomes; Biomarkers; High-Throughput Nucleotide Sequencing; Computational Biology; MicroRNAs; Gene Expression Profiling; Extracellular Vesicles
PubMed: 38928481
DOI: 10.3390/ijms25126761 -
International Journal of Molecular... Jun 2024The COVID-19 pandemic highlighted the need for a rapid, convenient, and scalable diagnostic method for detecting a novel pathogen amidst a global pandemic. While...
From SARS-CoV-2 to Global Preparedness: A Graphical Interface for Standardised High-Throughput Bioinformatics Analysis in Pandemic Scenarios and Surveillance of Drug Resistance.
The COVID-19 pandemic highlighted the need for a rapid, convenient, and scalable diagnostic method for detecting a novel pathogen amidst a global pandemic. While command-line interface tools offer automation for SARS-CoV-2 Oxford Nanopore Technology sequencing data analysis, they are inapplicable to users with limited programming skills. A solution is to establish such automated workflows within a graphical user interface software. We developed two workflows in the software Geneious Prime 2022.1.1, adapted for data obtained from the Midnight and Artic's nCoV-2019 sequencing protocols. Both workflows perform trimming, read mapping, consensus generation, and annotation on SARS-CoV-2 Nanopore sequencing data. Additionally, one workflow includes phylogenetic assignment using the bioinformatic tools pangolin and Nextclade as plugins. The basic workflow was validated in 2020, adhering to the requirements of the European Centre for Disease Prevention and Control for SARS-CoV-2 sequencing and analysis. The enhanced workflow, providing phylogenetic assignment, underwent validation at Uppsala University Hospital by analysing 96 clinical samples. It provided accurate diagnoses matching the original results of the basic workflow while also reducing manual clicks and analysis time. These bioinformatic workflows streamline SARS-CoV-2 Nanopore data analysis in Geneious Prime, saving time and manual work for operators lacking programming knowledge.
Topics: SARS-CoV-2; COVID-19; Humans; Computational Biology; Software; Phylogeny; Pandemics; Workflow; High-Throughput Nucleotide Sequencing; User-Computer Interface; Nanopore Sequencing
PubMed: 38928350
DOI: 10.3390/ijms25126645 -
International Journal of Molecular... Jun 2024The most significant genetic influence on eye color pigmentation is attributed to the intronic SNP rs12913832 in the gene, which interacts with the promoter region of...
The most significant genetic influence on eye color pigmentation is attributed to the intronic SNP rs12913832 in the gene, which interacts with the promoter region of the contiguous gene. This interaction, through the formation of a chromatin loop, modulates the transcriptional activity of , directly affecting eye color pigmentation. Recent advancements in technology have elucidated the precise spatial organization of the genome within the cell nucleus, with chromatin architecture playing a pivotal role in regulating various genome functions. In this study, we investigated the organization of the chromatin close to the locus in human lymphocyte nuclei using fluorescence in situ hybridization (FISH) and high-throughput chromosome conformation capture (Hi-C) data. The 3 Mb of genomic DNA that belonged to the chromosomal region 15q12-q13.1 revealed the presence of three contiguous chromatin loops, which exhibited a different level of compaction depending on the presence of the A or G allele in the SNP rs12913832. Moreover, the analysis of the genomic organization of the genes has demonstrated that this chromosomal region is evolutionarily highly conserved, as evidenced by the analysis of syntenic regions in species from other Vertebrate classes. Thus, the role of rs12913832 variant is relevant not only in determining the transcriptional activation of the gene but also in the chromatin compaction of a larger region, underscoring the critical role of chromatin organization in the proper regulation of the involved genes. It is crucial to consider the broader implications of this finding, especially regarding the potential regulatory role of similar polymorphisms located within intronic regions, which do not influence the same gene by modulating the splicing process, but they regulate the expression of adjacent genes. Therefore, caution should be exercised when utilizing whole-exome sequencing for diagnostic purposes, as intron sequences may provide valuable gene regulation information on the region where they reside. Thus, future research efforts should also be directed towards gaining a deeper understanding of the precise mechanisms underlying the role and mode of action of intronic SNPs in chromatin loop organization and transcriptional regulation.
Topics: Polymorphism, Single Nucleotide; Humans; Chromatin; Guanine Nucleotide Exchange Factors; Animals; Evolution, Molecular; Membrane Transport Proteins; In Situ Hybridization, Fluorescence; Vertebrates; Pigmentation; Ubiquitin-Protein Ligases
PubMed: 38928306
DOI: 10.3390/ijms25126602 -
International Journal of Molecular... Jun 2024Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the...
Paraformaldehyde (PFA) fixation is the preferred method for preserving tissue architecture for anatomical and pathological observations. Meanwhile, PFA reacts with the amine groups of biomolecules to form chemical cross-linking, which preserves RNA within the tissue. This has great prospects for RNA sequencing to characterize the molecular underpinnings after anatomical and pathological observations. However, RNA is inaccessible due to cross-linked adducts forming between RNA and other biomolecules in prolonged PFA-fixed tissue. It is also difficult to perform reverse transcription and PCR, resulting in low sequencing sensitivity and reduced reproducibility. Here, we developed a method to perform RNA sequencing in PFA-fixed tissue, which is easy to use, cost-effective, and allows efficient sample multiplexing. We employ cross-link reversal to recover RNA and library construction using random primers without artificial fragmentation. The yield and quality of recovered RNA significantly increased through our method, and sequencing quality metrics and detected genes did not show any major differences compared with matched fresh samples. Moreover, we applied our method for gene expression analysis in different regions of the mouse brain and identified unique gene expression profiles with varied functional implications. We also find significant dysregulation of genes involved in Alzheimer's disease (AD) pathogenesis within the medial septum (MS)/vertical diagonal band of Broca (VDB) of the 5×FAD mouse brain. Our method can thus increase the performance of high-throughput RNA sequencing with PFA-fixed samples and allows longitudinal studies of small tissue regions isolated by their in situ context.
Topics: Formaldehyde; Animals; Mice; Brain; Tissue Fixation; Sequence Analysis, RNA; Alzheimer Disease; Polymers; Gene Expression Profiling; High-Throughput Nucleotide Sequencing; RNA
PubMed: 38928210
DOI: 10.3390/ijms25126504 -
International Journal of Molecular... Jun 2024Mutations have driven the evolution and development of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with potential implications for...
Mutations have driven the evolution and development of new variants of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with potential implications for increased transmissibility, disease severity and vaccine escape among others. Genome sequencing is a technique that allows scientists to read the genetic code of an organism and has become a powerful tool for studying emerging infectious diseases. Here, we conducted a cross-sectional study in selected districts of the Eastern Province of Zambia, from November 2021 to February 2022. We analyzed SARS-CoV-2 samples ( = 76) using high-throughput sequencing. A total of 4097 mutations were identified in 69 SARS-CoV-2 genomes with 47% (1925/4097) of the mutations occurring in the spike protein. We identified 83 unique amino acid mutations in the spike protein of the seven Omicron sublineages (BA.1, BA.1.1, BA.1.14, BA.1.18, BA.1.21, BA.2, BA.2.23 and XT). Of these, 43.4% (36/83) were present in the receptor binding domain, while 14.5% (12/83) were in the receptor binding motif. While we identified a potential recombinant XT strain, the highly transmissible BA.2 sublineage was more predominant (40.8%). We observed the substitution of other variants with the Omicron strain in the Eastern Province. This work shows the importance of pandemic preparedness and the need to monitor disease in the general population.
Topics: Zambia; Humans; SARS-CoV-2; COVID-19; Genome, Viral; Spike Glycoprotein, Coronavirus; Mutation; Cross-Sectional Studies; Retrospective Studies; Phylogeny; Genomics; High-Throughput Nucleotide Sequencing
PubMed: 38928045
DOI: 10.3390/ijms25126338 -
International Journal of Molecular... Jun 2024Maturity-onset diabetes of the young (MODY) is part of the heterogeneous group of monogenic diabetes (MD) characterized by the non-immune dysfunction of pancreatic...
Maturity-onset diabetes of the young (MODY) is part of the heterogeneous group of monogenic diabetes (MD) characterized by the non-immune dysfunction of pancreatic β-cells. The diagnosis of MODY still remains a challenge for clinicians, with many cases being misdiagnosed as type 1 or type 2 diabetes mellitus (T1DM/T2DM), and over 80% of cases remaining undiagnosed. With the introduction of modern technologies, important progress has been made in deciphering the molecular mechanisms and heterogeneous etiology of MD, including MODY. The aim of our study was to identify genetic variants associated with MODY in a group of patients with early-onset diabetes/prediabetes in whom a form of MD was clinically suspected. Genetic testing, based on next-generation sequencing (NGS) technology, was carried out either in a targeted manner, using gene panels for monogenic diabetes, or by analyzing the entire exome (whole-exome sequencing). -MODY 2 was the most frequently detected variant, but rare forms of -MODY 13, specifically, -MODY 1, were also identified. We have emphasized the importance of genetic testing for early diagnosis, MODY subtype differentiation, and genetic counseling. We presented the genotype-phenotype correlations, especially related to the clinical evolution and personalized therapy, also emphasizing the particularities of each patient in the family context.
Topics: Humans; Diabetes Mellitus, Type 2; Genetic Testing; Male; Female; Genetic Counseling; Adult; Precision Medicine; High-Throughput Nucleotide Sequencing; Adolescent; Potassium Channels, Inwardly Rectifying; Young Adult; Child; Hepatocyte Nuclear Factor 4; Exome Sequencing; Genetic Predisposition to Disease; Mutation
PubMed: 38928025
DOI: 10.3390/ijms25126318 -
Genes Jun 2024The Mongolian gerbil is a distinctive experimental animal in China, as its genetic qualities possess significant value in the field of medical biology research. Here, we...
The Mongolian gerbil is a distinctive experimental animal in China, as its genetic qualities possess significant value in the field of medical biology research. Here, we aimed to establish an economical and efficient panel for genetic quality detection in Mongolian gerbils using single-nucleotide polymorphism (SNP) markers. To search for SNPs, we conducted whole-genome sequencing (WGS) in 40 Mongolian gerbils from outbred populations. Reliable screening criteria were established to preliminarily select SNPs with a wide genome distribution and high levels of polymorphism. Subsequently, a multiple-target regional capture detection system based on second-generation sequencing was developed for SNP genotyping. Based on the results of WGS, 219 SNPs were preliminarily selected, and they were established and optimized in a multiple-amplification system that included 206 SNP loci by genotyping three outbred populations. PopGen.32 analysis revealed that the average effective allele number, Shannon index, observed heterozygosity, expected heterozygosity, average heterozygosity, polymorphism information content, and other population genetic parameters of the Capital Medical University (CMU) gerbils were the highest, followed by those of Zhejiang gerbils and Dalian gerbils. Through scientific screening and optimization, we successfully established a novel, robust, and cost-effective genetic detection system for Mongolian gerbils by utilizing SNP markers for the first time.
Topics: Animals; Gerbillinae; Polymorphism, Single Nucleotide; Whole Genome Sequencing; Genotyping Techniques; Genetics, Population; China; Genotype; High-Throughput Nucleotide Sequencing
PubMed: 38927752
DOI: 10.3390/genes15060817 -
Genes Jun 2024Radiomics, an evolving paradigm in medical imaging, involves the quantitative analysis of tumor features and demonstrates promise in predicting treatment responses and... (Observational Study)
Observational Study
BACKGROUND
Radiomics, an evolving paradigm in medical imaging, involves the quantitative analysis of tumor features and demonstrates promise in predicting treatment responses and outcomes. This study aims to investigate the predictive capacity of radiomics for genetic alterations in non-small cell lung cancer (NSCLC).
METHODS
This exploratory, observational study integrated radiomic perspectives using computed tomography (CT) and genomic perspectives through next-generation sequencing (NGS) applied to liquid biopsies. Associations between radiomic features and genetic mutations were established using the Area Under the Receiver Operating Characteristic curve (AUC-ROC). Machine learning techniques, including Support Vector Machine (SVM) classification, aim to predict genetic mutations based on radiomic features. The prognostic impact of selected gene variants was assessed using Kaplan-Meier curves and Log-rank tests.
RESULTS
Sixty-six patients underwent screening, with fifty-seven being comprehensively characterized radiomically and genomically. Predominantly males (68.4%), adenocarcinoma was the prevalent histological type (73.7%). Disease staging is distributed across I/II (38.6%), III (31.6%), and IV (29.8%). Significant correlations were identified with mutations of p.Thr145Pro (shape_Sphericity), p.Arg167Gln (glszm_ZoneEntropy, firstorder_TotalEnergy), p.Asp2213Asn (glszm_GrayLevelVariance, firstorder_RootMeanSquared), and p.Asp1529Glu (glcm_Imc1). Patients with the p.Thr145Pro variant demonstrated markedly shorter median survival compared to the wild-type group (9.7 months vs. not reached, = 0.0143; HR: 5.35; 95% CI: 1.39-20.48).
CONCLUSIONS
The exploration of the intersection between radiomics and cancer genetics in NSCLC is not only feasible but also holds the potential to improve genetic predictions and enhance prognostic accuracy.
Topics: Humans; Carcinoma, Non-Small-Cell Lung; Male; Female; Lung Neoplasms; Middle Aged; High-Throughput Nucleotide Sequencing; Aged; Tomography, X-Ray Computed; Genomics; Mutation; Proto-Oncogene Proteins; Protein-Tyrosine Kinases; Prognosis; Adult; Anaplastic Lymphoma Kinase; Radiomics
PubMed: 38927739
DOI: 10.3390/genes15060803 -
Genes Jun 2024This study analyzed ancient DNA from the remains of horses unearthed from the Shihuyao tombs. These were found to date from the Han and Tang Dynasties in Xinjiang...
This study analyzed ancient DNA from the remains of horses unearthed from the Shihuyao tombs. These were found to date from the Han and Tang Dynasties in Xinjiang (approximately 2200 to 1100 years ago). Two high-quality mitochondrial genomes were acquired and analyzed using next-generation sequencing. The genomes were split into two maternal haplogroups, B and D, according to a study that included ancient and contemporary samples from Eurasia. A close genetic affinity was observed between the horse of the Tang Dynasty and Akhal-Teke horses according to the primitive horse haplotype G1. Historical evidence suggests that the ancient Silk Road had a vital role in their dissemination. Additionally, the matrilineal history of the Akhal-Teke horse was accessed and suggested that the early domestication of the breed was for military purposes.
Topics: Animals; Horses; Genome, Mitochondrial; China; DNA, Ancient; Haplotypes; DNA, Mitochondrial; Phylogeny; History, Ancient; High-Throughput Nucleotide Sequencing; Domestication
PubMed: 38927726
DOI: 10.3390/genes15060790 -
Genes Jun 2024Cardiomyopathies (CMs), one of the main causes of sudden death among the young population, are a heterogeneous group of myocardial diseases, usually with a genetic...
Cardiomyopathies (CMs), one of the main causes of sudden death among the young population, are a heterogeneous group of myocardial diseases, usually with a genetic cause. Next-Generation Sequencing (NGS) has expanded the genes studied for CMs; however, the yield is still around 50%. The systematic study of Copy Number Variants (CNVs) could contribute to improving our diagnostic capacity. These alterations have already been described as responsible for cardiomyopathies in some cases; however, their impact has been rarely assessed. We analyzed the clinical significance of CNVs in cardiomyopathies by studying 11,647 affected patients, many more than those considered in previously published studies. We evaluated the yield of the systematic study of CNVs in a production context using NGS and a novel CNV detection software tool v2.0 that has demonstrated great efficacy, maximizing sensitivity and avoiding false positives. We obtained a CNV analysis yield of 0.8% that fluctuated depending on the type of cardiomyopathy studied (0.29% HCM, 1.41% DCM, 1.88% ARVC, 1.8% LVNC, 1.45% RCM), and we present the frequency of occurrence for 18 genes that agglutinate the 95 pathogenic/likely pathogenic CNVs detected. We conclude the importance of including in diagnostic tests a systematic study of these genetic alterations for the different cardiomyopathies.
Topics: Humans; DNA Copy Number Variations; Cardiomyopathies; High-Throughput Nucleotide Sequencing; Male; Female; Adult; Clinical Relevance
PubMed: 38927710
DOI: 10.3390/genes15060774