Single Nucleotide Polymorphism Sites Research Articles

RNA barcode segments for SARS-CoV-2 identification from HCoVs and SARSr-CoV-2 lineages

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the pathogen responsible for coronavirus disease 2019 (COVID-19), continues to evolve, giving rise to more variants and global reinfections. Previous research has demonstrated that barcode segments can effectively and cost-efficiently identify specific species within closely related populations. In this study, we designed and tested RNA barcode segments based on genetic evolutionary relationships to facilitate the efficient and accurate identification of SARS-CoV-2 from extensive virus samples, including human coronaviruses (HCoVs) and SARSr-CoV-2 lineages. Nucleotide sequences sourced from NCBI and GISAID were meticulously selected and curated to construct training sets, encompassing 1733 complete genome sequences of HCoVs and SARSr-CoV-2 lineages. Through genetic-level species testing, we validated the accuracy and reliability of the barcode segments for identifying SARS-CoV-2. Subsequently, 75 main and subordinate species-specific barcode segments for SARS-CoV-2, located in ORF1ab, S, E, ORF7a, and N coding sequences, were intercepted and screened based on single-nucleotide polymorphism sites and weighted scores. Post-testing, these segments exhibited high recall rates (nearly 100%), specificity (almost 30% at the nucleotide level), and precision (100%) performance on identification. They were eventually visualized using one and two-dimensional combined barcodes and deposited in an online database (http://virusbarcodedatabase.top/). The successful integration of barcoding technology in SARS-CoV-2 identification provides valuable insights for future studies involving complete genome sequence polymorphism analysis. Moreover, this cost-effective and efficient identification approach also provides valuable reference for future research endeavors related to virus surveillance.

Development of molecular identification methods for Dryophytes suweonensis and D. japonicus, and their hybrids.

As hybridization can reduce biodiversity or cause extinction, it is important to identify both purebred parental species and their hybrids prior to conserving them. The Suwon tree frog, Dryophytes suweonensis, is an endangered wildlife species in Korea that shares its habitat and often hybridizes with the Japanese tree frog, D. japonicus. In particular, D. suweonensis, D. japonicus, and their hybrids often have abnormal ovaries and gonads, which are known causes that could threaten their existence. We collected 57 individuals from six localities where D. suweonensis is known to be present. High-resolution melting curve (HRM) analysis of the mitochondrial 12S ribosomal RNA gene was performed to determine the maternal species. Thereafter, the DNA sequences of five nuclear genes (SIAH, TYR, POMC, RAG1, and C-MYC) were analyzed to determine their parental species and hybrid status. The HRM analysis showed that the melting temperature of D. suweonensis was in the range of 79.0-79.3°C, and that of D. japonicus was 77.7-78.0°C, which clearly distinguished the two tree frog species. DNA sequencing of the five nuclear genes revealed 37 single-nucleotide polymorphism (SNP) sites, and STRUCTURE analysis showed a two-group structure as the most likely grouping solution. No heterozygous position in the purebred parental sequences with Q values ≥ 0.995 were found, which clearly distinguished the two treefrog species from their hybrids; 11 individuals were found to be D. suweonensis, eight were found to be D. japonicus, and the remaining 38 individuals were found to be hybrids. Thus, it was possible to unambiguously identify the parental species and their hybrids using HRM analysis and DNA sequencing methods. This study provided fundamental information for D. suweonensis conservation and restoration research.

Detection of single nucleotide polymorphisms based on triple-helix molecular switch combined with invader assay

Single-nucleotide polymorphisms (SNPs) can be relevant to disease susceptibility, disease pathogenesis, and efficacy of specific drugs. Monitoring SNPs plays a significant role in disease susceptibility and individual response to treatment, wherein optimal care can be provided to individuals based on their unique genetic and disease profiles. In this work, we have formulated a rapid and sensitive fluorescent biosensing platform based on a target-activated triple-helix molecular switch (THMS)-conformation-change-induced invader assay strategy for the detection of single nucleotide polymorphisms in the apolipoprotein E (APOE). The mutant DNA (MtDNA) of APOE gene hybridized with the THMS to form the three-dimensional invader structure over the SNP site. Subsequently, the specific structure would be recognized and cleaved by FEN1, leading to the recovery of FAM quenched by BHQ 1. Based on the high binding affinity of THMS to the target, our strategy could detect the MtDNA of AOPE gene without amplification at 0.27 nM. Furthermore, we achieved genotyping based on the difference in fluorescence signals and successfully applied this strategy to detect MtDNA of APOE gene in clinical samples, showing the great potential for monitoring various SNPs for biological research and disease diagnosis.

PIK3CA regulates development of diabetes retinopathy through the PI3K/Akt/mTOR pathway.

To explore their association with the development of diabetes retinopathy (DR), single nucleotide polymorphism (SNP) mutations were screened out by high-throughput sequencing and validated in patients diagnosed with DR. To understand the role of PIK3CA in the pathogenesis of DR and explore the relationship between PIK3CA,phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR),and DR, the effect of PIK3CA.rs17849079 mutation was investigated in a DR cell model. Twelve patients diagnosed with DR at the Qinghai Provincial People's Hospital from September 2020 to June 2021 were randomly selected as the case group, while 12 healthy subjects of similar age and gender who underwent physical examination in Qinghai Provincial People's Hospital physical examination center during the same period were randomly selected as the control group. Blood samples (2 mL) were collected from both groups using EDTA anticoagulant blood collection vessels and frozen at -20°C for future analysis. SNP mutations were detected by high-throughput sequencing, and the shortlisted candidates were subjected by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses. The detected SNP candidates were verified by expanding the sample size (first validation: 56 patients in the case group and 58 controls; second validation: 157 patients in the case group and 96 controls). A lentivirus vector carrying mutated or wild-type PIK3CA.rs17849079 was constructed. ARPE-19 cells were cultured in a medium supplemented with 10% fetal bovine serum (FBS) to establish a DR cell model. PIRES2-PIK3CA-MT and PIRES2-PIK3CA-WT vectors were transfected into DR model cells, which were categorized into control, mannitol, model, empty vector, PIK3CA wild-type, and PIK3CA mutant-type groups. Cell activity was detected by the cell counting kit (CCK)-8 assay, and cellular apoptosis was evaluated by flow cytometry. Glucose concentration and levels of cytokines tumor necrosis factor (TNF)-α and interleukin (IL)-1β were detected using enzyme-linked immunosorbent assay kits. The expression of PIK3CA, AKT1, mTOR, and VEGF genes was detected by real-time quantitative polymerase chain reaction (RT-qPCR), while the expression of PI3K, p-PI3K, AKT1, p-AKT1, mTOR, p-mTOR, and VEGF proteins was detected by western blotting. The mutated SNPs were mainly enriched in the PI3K/AKT pathway, calcium ion pathway, and glutamatergic synaptic and cholinergic synaptic signaling pathways. Seven SNPs, including PRKCE.rs1533476, DNAH11.rs10485983, ERAP1.rs149481, KLHL1.rs1318761, APOBEC3C.rs1969643, FYN.rs11963612, and KCTD1.rs7240205, were not related to the development of DR. PIK3CA.rs17849079 was prone to C/T mutation. The risk of DR increased with the presence of the C allele and decreased in the presence of the T allele. High glucose induced the expression of PIK3CA and VEGF mRNAs as well as the expression of PI3K, p-PI3K, p-AKT1, p-mTOR, and VEGF proteins in ARPE-19 cells, which led to secretion of inflammatory factors TNF-αand IL-1, cell apoptosis, and inhibition of cell proliferation. The PIK3CA.rs17849079 C allele accelerated the progression of DR. These biological effects were inhibited when the C allele of PIK3CA.rs17849079 was mutated to T allele. The mutated SNP sites in patients with DR were mainly enriched in PI3K/AKT, calcium ion, and glutamatergic synaptic and cholinergic synaptic signaling pathways. The rs17849079 allele of PIK3CA is prone to C/T mutation where the C allele increases the risk of DR. High glucose activates the expression of PIK3CA and promotes the phosphorylation of PI3K, which leads to the phosphorylation of AKT and mTOR. These effects consequently increase VEGF expression and accelerate the development of DR. The C to T allele mutation in PIK3CA.rs17849079 can play a protective role and reduce the risk of DR.

Deciphering complex antibiotic resistance patterns in Helicobacter pylori through whole genome sequencing and machine learning.

Helicobacter pylori (H.pylori, Hp) affects billions of people worldwide. However, the emerging resistance of Hp to antibiotics challenges the effectiveness of current treatments. Investigating the genotype-phenotype connection for Hp using next-generation sequencing could enhance our understanding of this resistance. In this study, we analyzed 52 Hp strains collected from various hospitals. The susceptibility of these strains to five antibiotics was assessed using the agar dilution assay. Whole-genome sequencing was then performed to screen the antimicrobial resistance (AMR) genotypes of these Hp strains. To model the relationship between drug resistance and genotype, we employed univariate statistical tests, unsupervised machine learning, and supervised machine learning techniques, including the development of support vector machine models. Our models for predicting Amoxicillin resistance demonstrated 66% sensitivity and 100% specificity, while those for Clarithromycin resistance showed 100% sensitivity and 100% specificity. These results outperformed the known resistance sites for Amoxicillin (A1834G) and Clarithromycin (A2147), which had sensitivities of 22.2% and 87%, and specificities of 100% and 96%, respectively. Our study demonstrates that predictive modeling using supervised learning algorithms with feature selection can yield diagnostic models with higher predictive power compared to models relying on single single-nucleotide polymorphism (SNP) sites. This approach significantly contributes to enhancing the precision and effectiveness of antibiotic treatment strategies for Hp infections. The application of whole-genome sequencing for Hp presents a promising pathway for advancing personalized medicine in this context.

Screening and analysis of single nucleotide polymorphismin the 3'-UTR microRNA target regions and its implications for lung tumorigenesis.

Aim: The study aims to identify high-impact single nucleotide polymorphisms (SNPs) in miRNA target sites of genes associated with lung cancer.Materials & methods: Lung cancer genes were obtained from Uniprot KB. miRNA target site SNPs were mined from MirSNP, miRdSNP and TargetScan. SNPs were shortlisted based on binding impact, minor allele frequencyand conservation. Gene expression was analyzed in genes with high-impact SNPs in healthy versus lung cancer tissue. Additionally, enrichment, pathwayand network analyzes were performed.Results: 19 high-impact SNPs were identified in miRNA target sites of lung cancer-associated genes. These SNPs affect miRNA binding and gene expression. The genes are involved in key cancerrelated pathways.Conclusion: The identified high-impact miRNA target site SNPs and associated genes provide a starting point for case-control studies in lung cancer patients in different populations.

Chromosomal-level genome assembly and single-nucleotide polymorphism sites of black-faced spoonbill Platalea minor.

Platalea minor, or black-faced spoonbill (Threskiornithidae), is a wading bird confined to coastal areas in East Asia. Due to habitat destruction, it was classified as globally endangered by the International Union for Conservation of Nature. However, the lack of genomic resources for this species hinders the understanding of its biology and diversity, and the development of conservation measures. Here, we report the first chromosomal-level genome assembly of P.minor using a combination of PacBio SMRT and Omni-C scaffolding technologies. The assembled genome (1.24Gb) contains 95.33% of the sequences anchored to 31 pseudomolecules. The genome assembly has high sequence continuity with scaffold length N50 = 53Mb. We predicted 18,780 protein-coding genes and measured high BUSCO score completeness (97.3%). Finally, we revealed 6,155,417 bi-allelic single nucleotide polymorphisms, accounting for ∼5% of the genome. This resource offers new opportunities for studying the black-faced spoonbill and developing conservation measures for this species.

Mitochondrial phylogeny and distribution of cytoplasmic male sterility-associated genes in Beta vulgaris.

Cytoplasmic male sterility (CMS) is a mitochondrial-encoded trait that confers reproductive defects in males but not in females or any vegetative function. Why CMS is so often found in plants should be investigated from the viewpoint of mitochondrial phylogeny. Beta vulgaris, including the wild subspecies maritima and cultivated subspecies vulgaris (e.g., sugar beet), is known to be mitochondrially polymorphic, from which multiple CMS mitochondria have been found, but their evolutionary relationship has been obscure. We first refined the B. vulgaris reference mitochondrial genome to conduct a more accurate phylogenetic study. We identified mitochondrial single-nucleotide polymorphic sites from 600 B. vulgaris accessions. Principal component analysis, hierarchical clustering analysis, and creation of a phylogenetic tree consistently suggested that B. vulgaris mitochondria can be classified into several groups whose geographical distribution tends to be biased toward either the Atlantic or Mediterranean coasts. We examined the distribution of CMS-associated mitochondrial genes from Owen, E- and G-type CMS mitochondria. About one-third of cultivated beets had Owen-type CMS, which reflects the prevalence of using Owen-type CMS in hybrid breeding. Occurrence frequencies for each of the three CMS genes in wild beet were less than 4%. CMS genes were tightly associated with specific mitochondrial groups that are phylogenetically distinct, suggesting their independent origin. However, homologous sequences of the Owen type CMS gene occurred in several different mitochondrial groups, for which an intricate explanation is necessary. Whereas the origin of cultivated beet had been presumed to be Greece, we found an absence of Owen-type mitochondria in Greek accessions.

Analysis of the Genetic Diversity in Tea Plant Germplasm in Fujian Province Based on Restriction Site-Associated DNA Sequencing.

Fujian province, an important tea-producing area in China, has abundant tea cultivars. To investigate the genetic relationships of tea plant cultivars in Fujian province and the characteristics of the tea plant varieties, a total of 70 tea cultivars from Fujian and other 12 provinces in China were subjected to restriction site-associated DNA sequencing (RAD-seq). A total of 60,258,975 single nucleotide polymorphism (SNP) sites were obtained. These 70 tea plant cultivars were divided into three groups based on analyzing the phylogenetic tree, principal component, and population structure. Selection pressure analysis indicated that nucleotide diversity was high in Southern China and genetically distinct from cultivars of Fujian tea plant cultivars, according to selection pressure analysis. The selected genes have significant enrichment in pathways associated with metabolism, photosynthesis, and respiration. There were ten characteristic volatiles screened by gas chromatography-mass spectrometry (GC-MS) coupled with multivariate statistical methods, among which the differences in the contents of methyl salicylate, 3-carene, cis-3-hexen-1-ol, (E)-4-hexen-1-ol, and 3-methylbutyraldehyde can be used as reference indicators of the geographical distribution of tea plants. Furthermore, a metabolome genome-wide association study (mGWAS) revealed that 438 candidate genes were related to the aroma metabolic pathway. Further analysis showed that 31 genes of all the selected genes were screened and revealed the reasons for the genetic differences in aroma among tea plant cultivars in Fujian and Southern China. These results reveal the genetic diversity in the Fujian tea plants as well as a theoretical basis for the conservation, development, and utilization of the Fujian highly aromatic tea plant cultivars.

Development of specific barcodes for identification of Aloe species based on chloroplast DNA barcoding

DNA barcoding is a widely used taxonomic tool for correctly identifying plant species. The chloroplast matK and rbcL are some of the most extensively studied variable coding genes of angiosperms and have been recommended as a universal barcode for terrestrial plants. Genus Aloe contains around 500 species, and most Aloe species have extensive traditional medicinal usage worldwide, viz., A. arborescens, A. ferox, A. maculate, and A. vera. This study evaluated the two chloroplast DNA barcodes (matK and rbcL) to develop a theoretical base for species identification and germplasm conservation of Aloe species. The Maximum Parsimony analysis was conducted to study the evolutionary relatedness of Aloe sequences using matK and rbcL sequences obtained from the NCBI nucleotide database. The results revealed that 49 Aloe accessions were segregated into four major clades using matK phylogenetic analysis. Likewise, the 29 Aloe accessions were allocated into two clades based on the rbcL phylogeny. Additionally, specific barcodes have been developed for the following species: A. vera, A. boylei, A. ciliaris, A. karasbergensis, and A. sinkatana based on SNP (Single Nucleotide Polymorphism) site analysis of barcode sequences and the corresponding DNA QR (quick response) code of each sequence was generated. This study provides a unique method for species identification of the genus Aloe and indicates the possibility of correctly identifying, discriminating, and documenting the Aloe species.

Circular intermediate-mediated horizontal transfer of the chromosome-encoded cfr(C) gene in multi-drug resistant Campylobacter coli from swine sources.

Campylobacter is a major zoonotic pathogen that causes gastrointestinal and, rarely, immune diseases in humans. The antimicrobial-resistance gene cfr(C) carried by Campylobacter and is a cfr-like gene that targets bacterial 23S rRNA through A2503 methylation. cfr(C) confers cross-resistance to five antimicrobial classes (PhLOPSA), including lincosamide, streptogramin A, and pleuromutilin, which are classified as critically important antimicrobials to human by the World Health Organization. To elucidate the genetic variation and horizontal transfer mechanism of cfr(C), we analyzed the genetic background and horizontal transfer unit of Campylobacter-derived cfr(C) through comparative genomic analysis. We identified nine cfr(C)-positive C. coli strains of 157 strains isolated from swine sources. Three novel cfr(C) gene single nucleotide polymorphism (SNP) sites (19delA, 674C > A, and 890 T > C) were identified from nine cfr(C)-positive strains. Among six identified cfr(C) SNP variant types (SNP-I to -VI), five types of randomly inserted cfr(C)-cassettes on chromosome and one type of plasmid-like element were identified, their gene cassette composition differing depending on the cfr(C) variants. Three of six cfr(C) cassette types contained aminoglycoside-streptothricin resistance cluster "aphA3-sat4-aadE." The cfr(C) gene cassette with pcp gene (GC-1, GC-4, and GC-5) formed a pcp-mediated circular intermediate "pcp-hp-cfr(C)-aphA3," which has not been previously reported. Other two cfr(C) cassette-types with ISChh1 formed circular intermediate "ISChh1-aphA3-cfr(C)-lnu (G)-pnp-ant1-hp-ATPase" and "ISChh1-aphA3-cfr(C)-hp." In conjugation assay, the pcp-mediated circular intermediate was naturally transferred to the plasmid of recipient C. coli wild-type strain from swine source, and comparative genomic analysis revealed that cfr(C) encoded in pcp-mediated circular intermediate was inserted into the plasmid of recipient by homologous recombination with pcp and aphA3. This study revealed that novel multidrug resistance gene cfr(C) carried by C. coli from swine sources can be highly genetically diverse and transferable. Moreover, we suggest that the transferability of chromosomal cfr(C) may contribute to the global spread of multidrug resistance against clinically important antimicrobials.

Nonadditive and allele-specific expression of ghrelin in hybrid tilapia.

Interspecies hybridization is an important breeding method to generate fishes with heterosis in aquaculture. Using this method, hybrid Nile tilapia (Oreochromis niloticus, ♀) × blue tilapia (Oreochromis aureus, ♂) has been produced and widely farmed due to its growth and appetite superiorities. However, the genetic mechanism of these advanced traits is still not well understood. Ghrelin is a crucial gene that regulates growth and appetite in fishes. In the present study, we focused on the expression characteristics and its regulation of ghrelin in the hybrid. The tissue distribution analysis showed that ghrelin was predominantly expressed in the stomach in the hybrid. Ghrelin was more highly expressed in the stomach in the hybrid and Nile tilapia, compared to blue tilapia, showing a nonadditive pattern. Two single-nucleotide polymorphism (SNP) sites were identified including T/C and C/G from the second exon in the ghrelin gene from Nile tilapia and blue tilapia. By pyrosequencing based on the SNP sites, the allele-specific expression (ASE) of ghrelin in the hybrid was assayed. The result indicated that ghrelin in the hybrid showed higher maternal allelic transcript ratios. Fasting significantly increased ghrelin overall expression at 4, 8, 12, 24, and 48h. In addition, higher maternal allelic transcript ratios were not changed in the fasting hybrids at 48h. The cis and trans effects were determined by evaluating the overall expression and ASE values in the hybrid. The expression of ghrelin was mediated by compensating cis and trans effects in hybrid. In summary, the present lines of evidence showed the nonadditive expression of ghrelin in the hybrid tilapia and its regulation by subgenomes, offering new insight into gene expression characteristics in hybrids.

Rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and real-time PCR.

Analyzing all biological evidence at a crime scene presents serious time, budget, and labor constraints. Therefore, selecting valid evidence is crucial for efficient screening. The ABO blood group is a marker that can serve as valid evidence for identifying investigative leads in criminal case. Conventional identification of ABO blood groups using serological methods has only been for blood and is difficult to apply to other body fluids. ABO genotyping was conducted by analyzing single nucleotide polymorphisms (SNP) representative of each blood group. However, this method is time-consuming, expensive, and requires sophisticated instruments. In this study, we developed rapid ABO genotyping method using loop-mediated isothermal amplification (LAMP) and multiplex real-time polymerase chain reaction (PCR). Three SNP sites in the ABO gene (nt 261, 526, and 803) were selected to classify the ABO genotypes. For the specificity test, we performed sequencing of 60 saliva samples to confirm that the genotyping. We conducted experiments to apply ABO genotyping using two amplification methods to mock forensic sample using cotton swab and filter paper. As a result, using LAMP, we successfully identified six ABO genotypes within 30min at a constant temperature (65 ℃). Moreover, by using multiple real-time PCR, it was possible to detect not only the major group but also the subgroup of the ABO genotype (ex. cis-AB). The amplification results using the new methods were in concordance with the sequencing results. Therefore, these ABO genotyping methods are expected to select valid evidence successfully and efficiently at the crime scene.

Genome-wide association study of growth traits and validation of key mutations (MSTN c.C861T) associated with the muscle mass of meat pigeons.

Selective breeding of meat pigeons is primarily based on growth traits, especially muscle mass (MM). Identification of functional genes and molecular markers of growth and slaughter traits through a genome-wide association study (GWAS) will help to elucidate the underlying molecular mechanisms and provide a theoretical basis for the selective breeding of meat pigeons. The phenotypic data of body weight (BW) and body size (BS) of 556 meat pigeons at 52 and 80 weeks of age were collected. In total, 160 434 high-quality single nucleotide polymorphism sites were obtained by restriction site-associated DNA sequencing. The GWAS analysis revealed that MSTN, IGF2BP3 and NCAPG/LCORL were important candidate genes affecting the growth traits of meat pigeons. IGF2BP3 and NCAPG/LCORL were highly correlated to BW and BS, which are related to overall growth and development, while MSTN was associated with pectoral thickness and BW. Phenotypic association validation with the use of two meat pigeon populations found that the MSTN mutation c.C861T determines the MM. These results provide new insights into the genetic mechanisms underlying phenotypic variations of growth traits and MM in commercial meat pigeons. The identified markers and genes provide a theoretical basis for the selective breeding of meat pigeons.

Genome-wide selective signatures mining the candidate genes for egg laying in goose

BackgroundImproving the egg production of goose is a crucial goal of breeding, because genetics is the key factor affecting egg production. Thus, we sequenced the genomes of 55 Chinese indigenous geese from six breeds, which were divided into the high egg-laying group (ZE, HY, and SC) and low egg-laying group (ZD, LH, and ST). Based on the results of the inter-population selection signal analysis, we mined the selected genome regions in the high egg-laying germplasm population to identify the key candidate genes affecting the egg-laying traits.ResultsAccording to the whole-genome sequencing data, the average sequencing depth reached 11.75X. The genetic relationships among those six goose breeds coincided with the breed’s geographical location. The six selective signal detection results revealed that the most selected regions were located on Chr2 and Chr12. In total, 12,051 single-nucleotide polymorphism (SNP) sites were selected in all six methods. Using the enrichment results of candidate genes, we detected some pathways involved in cell differentiation, proliferation, and female gonadal development that may cause differences in egg production. Examples of these pathways were the PI3K-Akt signaling pathway (IGF2, COMP, and FGFR4), animal organ morphogenesis (IGF2 and CDX4), and female gonad development (TGFB2).ConclusionOn analyzing the genetic background of six local goose breeds by using re-sequencing data, we found that the kinship was consistent with their geographic location. 107 egg-laying trait-associated candidate genes were mined through six selection signal analysis. Our study provides a critical reference for analyzing the molecular mechanism underlying differences in reproductive traits and molecular breeding of geese.

Prognostic Impact of Chromosomal Abnormalities in TP53-Mutated Acute Myeloid Leukemia and High-Risk Myelodysplastic Syndromes

Background: Background: Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) exhibit clinical heterogeneity, and genomic profiles play a crucial role in disease characteristics and survival outcomes. Elli Papaemmanuil et al. classified AML into 11 genomic subgroups (NEJM, 2016), where TP53 mutations and aneuploidy predicted dismal survival. Cases with TP53 mutations are often accompanied by complex chromosomal abnormalities (CAs), but the prognostic impact and target genes of CAs have not been fully studied. Methods: To clarify this issue, specimens of AML and MDS with excess blasts (MDS-EB) were collected from 1,001 patients enrolled at collaborating institutes of Kyoto Hematology Study Group between July 2017 and January 2023. Targeted capture sequencing with 1,216 single nucleotide polymorphism sites was used to analyze primary samples, allowing assessment of copy number alteration (CNA) profiles. The target gene panel included common mutations in myeloid neoplasms. Mutation calling was performed using the established pipeline Genomon 2. Prognostic impact of CNA, single nucleotide variants, and insertion/deletion with 10% or more frequency was evaluated, and the frequencies of CNA were calculated for the short and long arms of chromosomes 1-22 and the X chromosome. Univariate and multivariate analyses were conducted using statistical significance criteria of p < 0.05. Results: The average sequencing depth was 548. Among 1,001 patients, 224 (22.4%) harbored TP53 mutations. TP53-mutated cases had a median age of 70 (24-95) years old at diagnosis, with 117 (52.2%) being AML and 107 (47.8%) MDS-EB. TP53 biallelic mutation, defined by 2 or more mutations in TP53 or loss of heterogeneity of TP53 locus, was present in 200 (89.3%) of cases, more commonly associated with 3 or more CAs or CNA (complex karyotype (CK)-like) than monoallelic mutation (99% vs. 42%, p = 1.9x10 -13). The most frequent affected lesions were del(5q), del(7q), del(7p), del(12p), del(18q), gain(11q), and gain(21q) with frequencies of 76%, 59%, 34%, 33%, 28%, 27%, and 26%, respectively. In survival analysis, gain(21q), gain(19p), del(16q), del(3q), del(19p), del(13q), and del(4q), along with diagnosis of AML ( p = 2.0x10 -2), TP53 biallelic mutation ( p = 6.8x10 -3), and CK-like ( p = 3.4x10 -3), were associated with poor outcomes in univariate analysis. Multivariate analysis revealed gain(19p), del(3q), del(13q), del(4q), diagnosis of AML, and CK-like as independent poor prognostic factors. Here we have defined gain(19p), del(3q), del(13q), and del(4q) as “Risk CNA”. A significant difference in outcomes was observed between patients with and without Risk CNA (n = 110 and n = 114, respectively, Hazard ratio = 2.6, p = 6.0x10 -8): The 6-month survival rate was 70 (95% Confidence interval [60-79]) % for patients without Risk CNA and 42 [31-52] % for those with Risk CNA. Patients with two or more Risk CNAs had worse prognosis than those with one ( p = 2.7x10 -2), indicating independent or additive contributions to outcomes. Similar trends were observed when limiting the analysis to TP53-biallelic cases. Further investigation of Risk CNA target genes was performed by identifying commonly amplified/deleted lesions. Target genes of gain(19p) included DNM2, CARM1, SMARCA4, LDLR, and EPOR, while RB1 and TET2 were potential candidates of del(13q) and del(4q), respectively. Identification of candidate genes for del(3q) was challenging due to broad common deleted lesions. Conclusion: Chromosomal abnormalities stratify the prognosis of AML and MDS-EB with TP53 mutation. The number of Risk CNA also impacts survival. Investigation of target genes of Risk CNA suggests potential therapeutic targets for TP53-mutated myeloid neoplasms, warranting further study for treatment optimization.

Overlaps Between CDS Regions of Protein-Coding Genes in the Human Genome: A Case Study on the NR1D1-THRA Gene Pair.

For several decades, it has been known that a substantial number of genes within human DNA exhibit overlap; however, the biological and evolutionary significance of these overlaps remain poorly understood. This study focused on investigating specific instances of overlap where the overlapping DNA region encompasses the coding DNA sequences (CDSs) of protein-coding genes. The results revealed that proteins encoded by overlapping CDSs exhibit greater disorder than those from nonoverlapping CDSs. Additionally, these DNA regions were identified as GC-rich. This could be partially attributed to the absence of stop codons from two distinct reading frames rather than one. Furthermore, these regions were found to harbour fewer single-nucleotide polymorphism (SNP) sites, possibly due to constraints arising from the overlapping state where mutations could affect two genes simultaneously.While elucidating these properties, the NR1D1-THRA gene pair emerged as an exceptional case with highly structured proteins and a distinctly conserved sequence across eutherian mammals. Both NR1D1 and THRA are nuclear receptors lacking a ligand-binding domain at their C-terminus, which is the region where these gene pairs overlap. The NR1D1 gene is involved in the regulation of circadian rhythm, while the THRA gene encodes a thyroid hormone receptor, and both play crucial roles in various physiological processes. This study suggests that, in addition to their well-established functions, the specifically overlapping CDS regions of these genes may encode protein segments with additional, yet undiscovered, biological roles.

Development and genetic analysis of Tamarix chinensis single nucleotide polymorphism sites based on specific locus amplified fragment sequencing simplified genome technology

Development and genetic analysis of Tamarix chinensis single nucleotide polymorphism sites based on specific locus amplified fragment sequencing simplified genome technology

Molecular surveillance of chloroquine resistance in Plasmodium vivax isolates from malaria cases in Yunnan Province of China using pvcrt-o gene polymorphisms

BackgroundThe efficacy of chloroquine treatment for vivax malaria has been rarely evaluated due to a lack of an appropriate testing method. The objective of this study was to conduct molecular monitoring of chloroquine resistance in Plasmodium vivax strains from vivax malaria patients in Yunnan Province, focusing on the analysis of polymorphism in the P. vivax chloroquine resistance transporter protein orthologous gene (pvcrt-o).MethodsIn accordance with the principles of a cohort study, blood samples were collected from malaria cases diagnosed with a P. vivax mono-infection in Yunnan Province from 2020 to 2022. Segmental PCR was used to amplify the whole pvcrt-o gene in the blood samples and their products were subsequently sequenced. The sequencing data were arranged to obtain the full coding DNA sequence (CDS) as well as the gene’s promoter region sequences. The CDSs were aligned with the reference sequence (XM_001613407.1) of the P. vivax SalI isolate to identify the mutant loci.ResultsFrom a total of 375 blood samples taken from vivax malaria cases, 272 both whole gene CDSs (1272–1275 bp) and promoter DNA sequences (707 bp) of pvcrt-o gene were obtained. Among the whole CDSs, there were 7 single nucleotide polymorphic sites in which c.7 A>G was the minor allele frequency (MAF) site with 4.4% (12/272) detection rate. The mutation detection rate showed a significant decrease from 9.8% (10/102) in 2020 to 1.1% (1/92) in 2021 and 1.3% (1/78) in 2022, indicating statistical significance (χ2 = 11.256, P < 0.05). Among the identified 12 haplotypes, the majority of which were wild type (75.7%; 206/272). These four mutant haplotypes (Hap_3, Hap_5, Hap_9, and Hap_10) were classified as “K10 insertion type” and accounted for 12.1% (33/272). The detection rate of Hap_3 increased from 1.0% (1/102) in 2020 to 13.0% (12/92) in 2021 and 14.1% (11/78) in 2022, indicating statistical significance. A total of 23.8% (65/272) of the samples exhibited 14 bp (bp) deletions in the promoter region, occurring most frequently in the wild type haplotype (Hap_1) samples at a rate of 28.6% (59/206).ConclusionsIn recent years in Yunnan Province, a notable proportion of vivax malaria patients are infected by P. vivax strains with a “K10 insertion” and partial sequence deletions in the promoter region of the pvcrt-o gene, necessitating vigilance.

A FEN 1-driven DNA walker-like reaction coupling with magnetic bead-based separation for specific SNP detection.

Single-nucleotide polymorphism (SNP) plays a key role in the carcinogenesis of the human genome, and understanding the intrinsic relationship between individual genetic variations and carcinogenesis lies heavily in the establishment of a precise and sensitive SNP detection platform. Given this, a powerful and reliable SNP detection platform is proposed by a flap endonuclease 1 (FEN 1)-driven DNA walker-like reaction coupling with a magnetic bead (MB)-based separation. A carboxyfluorescein (FAM)-labeled downstream probe (DP) was decorated on a streptavidin magnetic bead (SMB). The target DNA, as a walker strand, was captured by hybridization with DP and an upstream probe (UP) to form a three-base overlapping structure and execute the walking function on the surface of SMB. FEN 1 was employed to specifically recognize the three-base overlapping structure and cut the 5'flap at the SNP site to report the walking event and signal amplification. Considering the fact that the fluorescence was labeled on the cleavage and uncleavage sequences of DP and the target DNA-triggered walking event was undistinguishable from the mixtures, magnetic separation came in handy for cleavage probe (CP) isolation and discrimination of the amplified signal from the background signal. In comparison with the conventional DNA walker reaction, this strategy was coupling with SMB-based separation, thus promising a powerful and reliable method for SNP detection and signal amplification.