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Genes Jun 2024Pathogenic variations in the gene have been detected with the development of next-generation sequencing (NGS)-based hereditary cancer panel testing technology. It also...
Pathogenic variations in the gene have been detected with the development of next-generation sequencing (NGS)-based hereditary cancer panel testing technology. It also reveals an increasing number of variants of uncertain significance (VUSs). Well-established functional tests are crucial to accurately reclassifying VUSs for effective diagnosis and treatment. We retrospectively analyzed the multi-gene cancer panel results of 922 individuals and performed in silico analysis following ClinVar classification. Then, we selected five breast cancer-diagnosed patients' missense VUSs (T1011R, T1104P/M1168K, R2027K, G2044A, and D2819) for reclassification. The effects of VUSs on BRCA2 function were analyzed using comet and H2AX phosphorylation (γH2AX) assays before and after the treatment of peripheral blood mononuclear cells (PBMCs) of subjects with the double-strand break (DSB) agent doxorubicin (Dox). Before and after Dox-induction, the amount of DNA in the comet tails was similar in VUS carriers; however, notable variations in γH2AX were observed, and according to combined computational and functional analyses, we reclassified T1001R as VUS-intermediate, T1104P/M1168K and D2819V as VUS (+), and R2027K and G2044A as likely benign. These findings highlight the importance of the variability of VUSs in response to DNA damage before and after Dox-induction and suggest that further investigation is needed to understand the underlying mechanisms.
Topics: Humans; Histones; Phosphorylation; Female; Breast Neoplasms; BRCA2 Protein; Comet Assay; High-Throughput Nucleotide Sequencing; Retrospective Studies; Mutation, Missense; DNA Breaks, Double-Stranded; DNA Damage
PubMed: 38927659
DOI: 10.3390/genes15060724 -
Genes May 2024Genetic counseling and treatment options for rare developmental disabilities (DDs) have been revolutionized by the opportunities made possible by using massively...
Genetic counseling and treatment options for rare developmental disabilities (DDs) have been revolutionized by the opportunities made possible by using massively parallel sequencing for diagnostic purposes [...].
Topics: Humans; Rare Diseases; Genetic Counseling; Phenotype; High-Throughput Nucleotide Sequencing; Genetic Testing; Developmental Disabilities
PubMed: 38927651
DOI: 10.3390/genes15060715 -
Genes May 2024The integration of target capture systems with next-generation sequencing has emerged as an efficient tool for exploring specific genetic regions with a high resolution...
The integration of target capture systems with next-generation sequencing has emerged as an efficient tool for exploring specific genetic regions with a high resolution and facilitating the rapid discovery of novel alleles. Despite these advancements, the application of targeted sequencing methodologies, such as the myBaits technology, in polyploid oat species remains relatively unexplored. In this study, we utilized the myBaits target capture method offered by Daicel Arbor Biosciences to detect variants and assess their reliability for variant detection in oat genomics and breeding. Ten oat genotypes were carefully chosen for targeted sequencing, focusing on specific regions on chromosome 2A to detect variants. The selected region harbors 98 genes. Precisely designed baits targeting the genes within these regions were employed for the target capture sequencing. We employed various mappers and variant callers to identify variants. After the identification of variants, we focused on the variants identified via all variants callers to assess the applicability of the myBaits sequencing methodology in oat breeding. In our efforts to validate the identified variants, we focused on two SNPs, one deletion and one insertion identified via all variant callers in the genotypes KF-318 and NOS 819111-70 but absent in the remaining eight genotypes. The Sanger sequencing of targeted SNPs failed to reproduce target capture data obtained through the myBaits technology. Similarly, the validation of deletion and insertion variants via high-resolution melting (HRM) curve analysis also failed to reproduce target capture data, again suggesting limitations in the reliability of the myBaits target capture sequencing using short-read sequencing for variant detection in the oat genome. This study shed light on the importance of exercising caution when employing the myBaits target capture strategy for variant detection in oats. This study provides valuable insights for breeders seeking to advance oat breeding efforts and marker development using myBaits target capture sequencing, emphasizing the significance of methodological sequencing considerations in oat genomics research.
Topics: Avena; High-Throughput Nucleotide Sequencing; Plant Breeding; Polymorphism, Single Nucleotide; Genome, Plant; Genomics; Genotype; Sequence Analysis, DNA
PubMed: 38927635
DOI: 10.3390/genes15060700 -
Biomolecules May 2024Normal testicular development ensures the process of spermatogenesis, which is a complex biological process. The sustained high productivity of spermatogenesis...
Normal testicular development ensures the process of spermatogenesis, which is a complex biological process. The sustained high productivity of spermatogenesis throughout life is predominantly attributable to the constant proliferation and differentiation of spermatogonial stem cells (SSCs). The self-renewal and differentiation processes of SSCs are strictly regulated by the SSC niche. Therefore, understanding the developmental pattern of SSCs is crucial for spermatogenesis. The Shaziling pig is a medium-sized indigenous pig breed originating from central China. It is renowned for its superior meat quality and early male sexual maturity. The spermatogenic ability of the boars is of great economic importance to the pig industry. To investigate testicular development, particularly the pattern of SSC development in Shaziling pigs, we used single-cell transcriptomics to identify gene expression patterns in 82,027 individual cells from nine Shaziling pig testes at three key postnatal developmental stages. We generated an unbiased cell developmental atlas of Shaziling pig testicular tissues. We elucidated the complex processes involved in the development of SSCs within their niche in the Shaziling pig. Specifically, we identified potential marker genes and cellular signaling pathways that regulate SSC self-renewal and maintenance. Additionally, we proposed potential novel marker genes for SSCs that could be used for SSC isolation and sorting in Shaziling pigs. Furthermore, by immunofluorescence staining of testicular tissues of different developmental ages using marker proteins (UCHL1 and KIT), the developmental pattern of the spermatogonia of Shaziling pigs was intensively studied. Our research enhances the comprehension of the development of SSCs and provides a valuable reference for breeding Shaziling pigs.
Topics: Animals; Male; Swine; RNA-Seq; Spermatogonia; Testis; Adult Germline Stem Cells; Single-Cell Analysis; Cell Differentiation; Spermatogenesis; Stem Cells; Transcriptome
PubMed: 38927011
DOI: 10.3390/biom14060607 -
Zhongguo Shi Yan Xue Ye Xue Za Zhi Jun 2024To analyze the DTA (, , ) mutations in patients with myeloproliferative neoplasms (MPN), and preliminarily explore their correlation with thromboembolism.
OBJECTIVE
To analyze the DTA (, , ) mutations in patients with myeloproliferative neoplasms (MPN), and preliminarily explore their correlation with thromboembolism.
METHODS
Clinical characteristics of 62 patients diagnosed de novo MPN at Central Hospital Affiliated to Shandong First Medical University from September 2016 to September 2022 were retrospectively analyzed. Next-generation sequencing was used to detect 35 MPN-related genes, and the DTA mutations in MPN patients and their relationship with thromboembolic events were analyzed.
RESULTS
75.8% (47/62) of the patients presented pathogenic non-driver mutations, and the mean number of pathogenic non-driver mutations per patient was 1.08. Among them, the most frequently mutated non-driver genes were (38.7%, 24/62), (9.7%, 6/62) and (6.5%, 4/62). The presence of DTA gene mutations was 50% (31/62) in the total MPN patients, and mainly accompanied by driver mutations. The mutation rate of DTA in patients aged ≥60 years was significantly higher than that in patients <60 years old ( =0.039). The incidence of thromboembolism in patients with DTA mutation was 58.1% (18/31), which was significantly higher than that in patients without DTA mutation (19.4%, 6/31) ( =0.002). The gene mutation rate in MPN patients with thromboembolism was 66.7% (16/24), which was significantly higher than that in patients without thromboembolism (21.1%, 8/38) ( =0.00).
CONCLUSION
Patients with MPN have a higher incidence of DTA mutations, which are mainly accompanied by driver gene mutations. The incidence of thromboembolism in MPN patients with DTA mutations is higher than that in patients without DTA mutations. Especially, the elderly (≥60 years) essential thrombocythemia(ET) and polycythemia vera(PV) patients with mutation should be vigilant for thromboembolic events.
Topics: Humans; Mutation; Dioxygenases; Middle Aged; Myeloproliferative Disorders; Thromboembolism; Retrospective Studies; Proto-Oncogene Proteins; DNA-Binding Proteins; Repressor Proteins; DNA Methyltransferase 3A; DNA (Cytosine-5-)-Methyltransferases; Male; Female; High-Throughput Nucleotide Sequencing
PubMed: 38926973
DOI: 10.19746/j.cnki.issn.1009-2137.2024.03.025 -
BMC Infectious Diseases Jun 2024Malassezia restricta, a lipophilic and lipodependent yeast belonging to the basidiomycetes group, is an opportunistic fungal pathogen associated with various skin... (Review)
Review
BACKGROUND
Malassezia restricta, a lipophilic and lipodependent yeast belonging to the basidiomycetes group, is an opportunistic fungal pathogen associated with various skin diseases, including seborrheic dermatitis and dandruff. Typically, Malassezia infection in neonates manifests as fungemia or hematogenous dissemination to the bone or lungs. However, vertebral osteomyelitis caused by these fungi is rarely reported owing to non-specific clinical presentations and laboratory/imaging findings. The Pathogen Metagenomics Sequencing (PMseq) technique enables direct high-throughput sequencing of infected specimens, facilitating the rapid and accurate detection of all microorganisms in clinical samples through comprehensive reports.
CASE PRESENTATION
A 52-year-old male was admitted to our hospital on July 20, 2022 with a 3-month history of ambulatory difficulties and localized low back pain. Magnetic Resonance Imaging (MRI) examination of the spinal column revealed irregular bone destruction affecting the L2, L3, and L5 vertebral bodies. Additionally, low T1 and high T2 intensity lesions were observed at the intervertebral discs between L3 and L5. The presumptive diagnosis of tuberculous spondylitis was made based on the imaging findings, despite negative results in all mycobacterium tests. However, the patient exhibited no improvement after receiving regular anti-tuberculosis treatment for 3 months. Subsequent MRI revealed an expansive abnormal signal within the vertebral body, leading to progressive bone destruction. The absence of spinal tuberculosis or other infective microorganisms was confirmed through culture from blood and pathological tissue from the L4 vertebral body. Subsequently, PMseq was performed on the specimens, revealing M. restricta as the predominant pathogen with the highest relative abundance value. The pathological examination revealed the presence of fungal mycelium in the L4 vertebral body, with positive findings on periodic Schiff-methenamine and periodic acid-Schiff staining. The anti-tuberculosis treatment was discontinued, and an antifungal combination of fluconazole and voriconazole was administered. All symptoms were resolved after 7 consecutive months of treatment, and the patient was able to ambulate autonomously. Vertebral lesions were reduced on MRI during the 13-month follow-up.
CONCLUSIONS
M. restricta is not a commonly recognized pathogen associated with infectious vertebral osteomyelitis. However, PMseq can aid in diagnosis, timely treatment, and decision making for some non-specific infectious diseases.
Topics: Humans; Male; Osteomyelitis; Middle Aged; Malassezia; Metagenomics; Magnetic Resonance Imaging; Antifungal Agents; High-Throughput Nucleotide Sequencing
PubMed: 38926679
DOI: 10.1186/s12879-024-09512-9 -
BMC Plant Biology Jun 2024During male gametogenesis of flowering plants, sperm cell lineage (microspores, generative cells, and sperm cells) differentiated from somatic cells and acquired...
BACKGROUND
During male gametogenesis of flowering plants, sperm cell lineage (microspores, generative cells, and sperm cells) differentiated from somatic cells and acquired different cell fates. Trimethylation of histone H3 on lysine 4 (H3K4me3) epigenetically contributes to this process, however, it remained unclear how H3K4me3 influences the gene expression in each cell type. Here, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) to obtain a genome-wide landscape of H3K4me3 during sperm cell lineage development in tomato (Solanum lycopersicum).
RESULTS
We show that H3K4me3 peaks were mainly enriched in the promoter regions, and intergenic H3K4me3 peaks expanded as sperm cell lineage differentiated from somatic cells. H3K4me3 was generally positively associated with transcript abundance and served as a better indicator of gene expression in somatic and vegetative cells, compared to sperm cell lineage. H3K4me3 was mutually exclusive with DNA methylation at 3' proximal of the transcription start sites. The microspore maintained the H3K4me3 features of somatic cells, while generative cells and sperm cells shared an almost identical H3K4me3 pattern which differed from that of the vegetative cell. After microspore division, significant loss of H3K4me3 in genes related to brassinosteroid and cytokinin signaling was observed in generative cells and vegetative cells, respectively.
CONCLUSIONS
Our results suggest the asymmetric division of the microspore significantly reshapes the genome-wide distribution of H3K4me3. Selective loss of H3K4me3 in genes related to hormone signaling may contribute to functional differentiation of sperm cell lineage. This work provides new resource data for the epigenetic studies of gametogenesis in plants.
Topics: Solanum lycopersicum; Histones; Cell Lineage; Genome, Plant; DNA Methylation; Gene Expression Regulation, Plant; Pollen; Epigenesis, Genetic; Chromatin Immunoprecipitation Sequencing
PubMed: 38926660
DOI: 10.1186/s12870-024-05318-8 -
Scientific Reports Jun 2024Viral infections pose significant public health challenges, exemplified by the global impact of COVID-19 caused by SARS-CoV-2. Understanding the intricate molecular...
Viral infections pose significant public health challenges, exemplified by the global impact of COVID-19 caused by SARS-CoV-2. Understanding the intricate molecular mechanisms governing virus-host interactions is pivotal for effective intervention strategies. Despite the burgeoning multi-omics data on viral infections, a centralized database elucidating host responses to viruses remains lacking. In response, we have developed a comprehensive database named 'MOI' (available at http://www.fynn-guo.cn/ ), specifically designed to aggregate processed Multi-Omics data related to viral Infections. This meticulously curated database serves as a valuable resource for conducting detailed investigations into virus-host interactions. Leveraging high-throughput sequencing data and metadata from PubMed and Gene Expression Omnibus (GEO), MOI comprises over 3200 viral-infected samples, encompassing human and murine infections. Standardized processing pipelines ensure data integrity, including bulk RNA sequencing (RNA-seq), single-cell RNA-seq (scRNA-seq), Chromatin Immunoprecipitation sequencing (ChIP-seq), and Assay for Transposase-Accessible Chromatin using sequencing (ATAC-seq). MOI offers user-friendly interfaces presenting comprehensive cell marker tables, gene expression data, and epigenetic landscape charts. Analytical tools for DNA sequence conversion, FPKM calculation, differential gene expression, and Gene Ontology (GO)/ Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment enhance data interpretation. Additionally, MOI provides 16 visualization plots for intuitive data exploration. In summary, MOI serves as a valuable repository for researchers investigating virus-host interactions. By centralizing and facilitating access to multi-omics data, MOI aims to advance our understanding of viral pathogenesis and expedite the development of therapeutic interventions.
Topics: Humans; Animals; Mice; COVID-19; SARS-CoV-2; Databases, Genetic; Virus Diseases; High-Throughput Nucleotide Sequencing; Host-Pathogen Interactions; Chromatin Immunoprecipitation Sequencing; Multiomics
PubMed: 38926513
DOI: 10.1038/s41598-024-65629-6 -
Scientific Reports Jun 2024Hypervariable region sequencing of the 16S ribosomal RNA (rRNA) gene plays a critical role in microbial ecology by offering insights into bacterial communities within...
Hypervariable region sequencing of the 16S ribosomal RNA (rRNA) gene plays a critical role in microbial ecology by offering insights into bacterial communities within specific niches. While providing valuable genus-level information, its reliance on data from targeted genetic regions limits its overall utility. Recent advances in sequencing technologies have enabled characterisation of the full-length 16S rRNA gene, enhancing species-level classification. Although current short-read platforms are cost-effective and precise, they lack full-length 16S rRNA amplicon sequencing capability. This study aimed to evaluate the feasibility of a modified 150 bp paired-end full-length 16S rRNA amplicon short-read sequencing technique on the Illumina iSeq 100 and 16S rRNA amplicon assembly workflow by utilising a standard mock microbial community and subsequently performing exploratory characterisation of captive (zoo) and free-ranging African elephant (Loxodonta africana) respiratory microbiota. Our findings demonstrate that, despite generating assembled amplicons averaging 869 bp in length, this sequencing technique provides taxonomic assignments consistent with the theoretical composition of the mock community and respiratory microbiota of other mammals. Tentative bacterial signatures, potentially representing distinct respiratory tract compartments (trunk and lower respiratory tract) were visually identified, necessitating further investigation to gain deeper insights into their implication for elephant physiology and health.
Topics: Animals; Elephants; RNA, Ribosomal, 16S; Bacteria; Microbiota; High-Throughput Nucleotide Sequencing; Respiratory System; Animals, Zoo; Sequence Analysis, DNA; Animals, Wild; Phylogeny
PubMed: 38926469
DOI: 10.1038/s41598-024-65841-4 -
Methods in Molecular Biology (Clifton,... 2024In traditional cell line design pipelines, cost- and time-intensive long-term stability studies must be performed due to random integration of the transgene into the...
In traditional cell line design pipelines, cost- and time-intensive long-term stability studies must be performed due to random integration of the transgene into the genome. By this, integration into epigenetically silenced regions can lead to silencing of the recombinant promoter over time. Site-specific integration into regions with active chromatin structure can overcome this problem and lead to strong and stable gene expression. Here, we describe a detailed protocol to identify integration sites with epigenetically preferable properties by chromatin immunoprecipitation sequencing and use them for stable and strong gene expression by applying CRISPR/Cas9. Furthermore, the examination of the integration sites with focus on Cas9-targeted sequencing with nanopores is described.
Topics: CRISPR-Cas Systems; Humans; Histone Code; Gene Editing; Cell Line; Epigenesis, Genetic; Chromatin Immunoprecipitation Sequencing; Histones; Chromatin
PubMed: 38926282
DOI: 10.1007/978-1-0716-3878-1_14