Single Nucleotide Research Articles

Complete chloroplast genome of Gentianopsis barbata and comparative analysis with related species from Gentianaceae.

Gentianopsis barbata is an essential medicinal plant in China with high ornamental and medicinal values. Unfortunately, the study of the chloroplast genome of this plant still has a gap. This study sequenced and characterized the complete chloroplast genome of G. barbata. The complete chloroplast genome of G. barbata is a typical circular structure of 151 123bp. It consists of a large single-copy region (82 690bp) and a small single-copy region (17 887bp) separated by a pair of inverted repeats (25 273bp), which covers 78 protein-coding genes, 30 tRNAs, and 4 rRNAs. The long repeat sequence analysis showed that the P-type (palindromic) sequences were the major long repeat sequences. Thirty-seven simple sequence repeats were identified, most of which were single nucleotides. The Bayesian inference tree, maximum likelihood tree, and neighbor-joining tree suggested that G. barbata is grouped with Gentianopsis grandis and Gentianopsis paludosa. The divergence time analysis showed that G. barbata diverged at 1.243 Mya. Comparative analysis of chloroplast genomes can reveal interspecific diversity, and regions with high variation can be used to develop molecular markers applicable to various research areas. Our results provide a new insight into plastome evolution and a valuable resource for further studies on G. barbata.

Kinetic Analysis of T4 Polynucleotide Kinase via Isothermal Titration Calorimetry

Isothermal titration calorimetry (ITC) measures the heat absorbed or released from biochemical processes. Although the primary application of ITC has been in binding studies, the reaction enthalpy detected by ITC can be used as a probe to quantify enzyme activity. Kinases are an important class of enzymes that are involved in many types of cellular regulation, but kinetic studies of kinases by ITC have been limited. T4 polynucleotide kinase (T4 PNK) is proposed as a model system to optimize and validate the ITC analysis conditions for a kinase. T4 PNK typically phosphorylates an oligonucleotide, but a single nucleotide can serve as a substrate if a 3′‐phosphate group is present. We used ITC to study the T4 PNK‐catalyzed conversion of 3′‐AMP to adenosine‐3′,5′‐bisphosphate. To initiate the reaction, a single injection of 3′‐AMP substrate was made into the ITC cell containing T4 PNK and ATP at pH 7.6 and 30oC. The shape of the resultant Differential Power versus Time curve is determined by the kinetic properties of the enzyme. Integration of the area under the curve yields reaction enthalpy. The apparent ∆H for the reaction was found to vary depending on the type of buffer used. This result was explained by differential protonation of reactants and products at pH 7.6, and indicated the release of approximately 0.6 protons per mole for the forward reaction. Extrapolation was used to estimate an intrinsic (i.e. buffer‐independent) ∆H of ‐25 kJ/mol. The Michaelis‐Menten constant, KM, was 1.3E‐05 M, and the turnover number, kcat, was 8/sec. The reaction enthalpy and kinetic parameters for T4 PNK phosphorylation of 3′‐AMP were assessed successfully by single injection ITC analysis. This finding supports the potential to analyze more complex kinase systems using ITC.

Bioinformatic Analyzes of the Association Between Upregulated Expression of JUN Gene via APOBEC-Induced FLG Gene Mutation and Prognosis of Cervical Cancer

Globally, cervical cancer (CC) is the most common malignant tumor of the female reproductive system and its incidence is only second after breast cancer. Although screening and advanced treatment strategies have improved the rates of survival, some patients with CC still die due to metastasis and drug resistance. It is considered that cancer is driven by somatic mutations, such as single nucleotide, small insertions/deletions, copy number, and structural variations, as well as epigenetic changes. Previous studies have shown that cervical intraepithelial neoplasia is associated with copy number variants (CNVs) and/or mutations in cancer-related genes. Further, CC is also related to genetic mutations. The present study analyzed the data on somatic mutations of cervical squamous cell carcinoma (CESC) in the Cancer Genome Atlas database. It was evident that the Apolipoprotein B mRNA editing enzyme-catalyzed polypeptide-like (APOBEC)-related mutation of the FLG gene can upregulate the expression of the JUN gene and ultimately lead to poor prognosis for patients with CC. Therefore, the findings of the current study provide a new direction for future treatment of CC.

Mutation of rpsL Gene in Streptomycin-Resistant Pseudomonas syringae pv. actinidiae Biovar 3 Strains Isolated from Korea

Pseudomonas syringae pv. actinidiae (Psa) is the causal agent responsible for the bacterial canker disease of kiwifruit plants. Psa strains are divided into five different biovars based on genetic and biochemical characteristics. Among them, biovar 2 and 3 strains of Psa were isolated and have been causing widespread damages in Korea. One of the most effective ways to control Psa is to use an antibiotic such as streptomycin. However, Psa strains resistant to this antibiotic were isolated in Korea, and an earlier study revealed that the resistance in the biovar 2 is associated with strA-strB genes. This study aimed to determine the molecular resistance mechanism of Psa biovar 3 strains to streptomycin. Sequencing the rpsL gene encoding ribosomal protein S12 from three streptomycin-resistant strains screened in the laboratory revealed that a spontaneous mutation occurred either at codon 43 or 88. Meanwhile, in four streptomycin-resistant strains of Psa biovar 3 isolated from two kiwifruit orchards, a single nucleotide in codon 43 of the rpsL, which is AAA in streptomycin-sensitive strain, was substituted for AGA causing an amino acid change from lysine to arginine. The resistant mechanism in all biovar 3 strains obtained in Korea was identified as a mutation of the rpsL gene.

Association of CYP19 Gene Polymorphism with Breast Cancer in Iraqi Women

The basic enzyme in estradiol synthesis is, Aromatase, it is encoded by the gene called CYP19A1. Given the importance of estrogen in the development of Breast cancer risk, we studyt genetic polymorphisms in the CYP19A1 gene the, including rs10046, in a group of 45 patient women in Kerbala province, Iraq. Our aim to investigate CYP19AI polymorphism its impact on patients of Breast cancer in Iraqi women. We found in our study, that in Iraqi postmenopausal women,( rs10046) single nucleotides and polymorphism (SNPs) was prognostic of different levels of estrone and Estradiol. Depending on the BMI scale reveals 22.25% with the breast cancer group in Comparison with 30% of healthy women. In case of BMI with( 20-24) in the breast cancer group were 22.2% compared with the healthy group 20%., While BMI of >24 we found 33.4% in case of Breast cancer affected group in comparison with 20% of the unaffected group (Healthy). While in case of BMI >30 there were 22.2% in case of the Breast cancer group in comparison with 30% of unaffected (healthy) group. The molecular analysis of CYP19 gene ,reveals that A allele frequency was (0.6333) and G allele (0.3667) upon that, we found that the number of patients which have had breast cancer were 28 and 17 ,respectively. In summary, our data show that polymorphisms in the CYP19A1 gene may contribute to Breast carcinogenesis in Iraqi women.

Novel Pathogenic Sequence Variation m.5789T>C Causes NARP Syndrome and Promotes Formation of Deletions of the Mitochondrial Genome.

Background and ObjectivesWe report the pathogenic sequence variant m.5789T>C in the anticodon stem of the mitochondrial tRNA for cysteine as a novel cause of neuropathy, ataxia, and retinitis pigmentosa (NARP), which is usually associated with pathogenic variants in the MT-ATP6 gene.MethodsTo address the correlation of oxidative phosphorylation deficiency with mutation loads, we performed genotyping on single laser-dissected skeletal muscle fibers. Stability of the mitochondrial tRNACys was investigated by Northern blotting. Accompanying deletions of the mitochondrial genome were detected by long-range PCR and their breakpoints were determined by sequencing of single-molecule amplicons.ResultsThe sequence variant m.5789T>C, originating from the patient's mother, decreases the stability of the mitochondrial tRNA for cysteine by disrupting the anticodon stem, which subsequently leads to a combined oxidative phosphorylation deficiency. In parallel, we observed a prominent cluster of low-abundance somatic deletions with breakpoints in the immediate vicinity of the m.5789T>C variant. Strikingly, all deletion-carrying mitochondrial DNA (mtDNA) species, in which the corresponding nucleotide position was not removed, harbored the mutant allele, and none carried the wild-type allele.DiscussionIn addition to providing evidence for the novel association of a tRNA sequence alteration with NARP syndrome, our observations support the hypothesis that single nucleotide changes can lead to increased occurrence of site-specific mtDNA deletions through the formation of an imperfect repeat. This finding might be relevant for understanding mechanisms of deletion generation in the human mitochondrial genome.

Suppressor TRNA-mediated rescue of hERG LQTS2 nonsense mutations

Congenital long QT syndrome (LQTS), characterized by prolongation of the ventricular action potential duration and an extended QT interval on the electrocardiogram, increases the propensity for lethal ventricular arrythmias and sudden cardiac death. In particular, LQTS type 2 (LQT2) results from deleterious loss-of-function mutations in the human ether-à-go-go related gene (hERG), which encodes for the pore-forming subunit of a voltage-dependent potassium channel in cardiac cells. LQTS2-associated nonsense mutations in hERG are characterized by single nucleotide changes which convert an amino acid-encoding codon into one of the three protein translation stop codons.

Identification of the novel allele, HLA-A*01:01:01:92, in an individual from North India.

HLA-A*01:01:01:92 differs from HLA-A*01:01:01:01 by a single nucleotide G->C change at gDNA-56 position.

In Silico Analysis of Collagens Missense SNPs and Human Abnormalities.

Collagens are the most abundant proteins in the extra cellular matrix/ECM of human tissues that are encoded by different genes. There are single nucleotide polymorphisms/SNPs which are considered as the most useful biomarkers for some disease diagnosis or prognosis. The aim of this study is screening and identifying the functional missense SNPs of human ECM-collagens and investigating their correlation with human abnormalities. All of the missense SNPs were retrieved from the NCBI SNP database and screened for a global frequency of more than 0.1. Seventy missense SNPs that met the screening criteria were characterized for functional and stability impact using six and three protein analysis tools, respectively. Next, HOPE and geneMANIA analysis tools were used to show the effect of SNPs on three-dimensional structure (3D) and physical interaction of proteins. Results showed that 13 missense SNPs (rs2070739, rs28381984, rs13424243, rs1800517, rs73868680, rs12488457, rs1353613, rs59021909, rs9830253, rs2228547, rs3753841, rs2855430, and rs970547), which are in nine different collagen genes, affect the structure and function of different collagen proteins. Among these polymorphisms, COL4A3-rs13424243 and COL6A6-rs59021909 were predicted as the most effective ones. On the other hand, designed mutated and native 3D of rs13424243 variant illustrated that it can disturb the protein motifs. Also, geneMANIA predicted that COL4A3 and COL6A6 are interacting with some proteins including: DDR1, COL6A1, COL11A2 and so on. Based on our findings, ECM-collagens functional SNPs are important and may be considered as a risk factor or molecular marker for human disorders in the future studies.

Customization of a DADA2-based pipeline for fungal internal transcribed spacer 1 (ITS1) amplicon data sets.

Identification and analysis of fungal communities commonly rely on internal transcribed spacer–based (ITS-based) amplicon sequencing. There is no gold standard used to infer and classify fungal constituents since methodologies have been adapted from analyses of bacterial communities. To achieve high-resolution inference of fungal constituents, we customized a DADA2-based pipeline using a mix of 11 medically relevant fungi. While DADA2 allowed the discrimination of ITS1 sequences differing by single nucleotides, quality filtering, sequencing bias, and database selection were identified as key variables determining the accuracy of sample inference. Due to species-specific differences in sequencing quality, default filtering settings removed most reads that originated from Aspergillus species, Saccharomyces cerevisiae, and Candida glabrata. By fine-tuning the quality filtering process, we achieved an improved representation of the fungal communities. By adapting a wobble nucleotide in the ITS1 forward primer region, we further increased the yield of S. cerevisiae and C. glabrata sequences. Finally, we showed that a BLAST-based algorithm based on the UNITE+INSD or the NCBI NT database achieved a higher reliability in species-level taxonomic annotation compared with the naive Bayesian classifier implemented in DADA2. These steps optimized a robust fungal ITS1 sequencing pipeline that, in most instances, enabled species-level assignment of community members.

Genetic basis of cardiovascular aging is at the core of human longevity

Aging is an archetypical complex process influenced by genetic and environmental factors. Genetic variants impart a gradient of effect sizes, albeit the effect sizes seem to be skewed toward those with small effect sizes. On one end of the spectrum are the rare monogenic premature aging syndromes, such as Hutchinson Gilford Progeria Syndrome, whereby single nucleotide changes lead to rapidly progressive premature aging. On the end of the spectrum is the complex, slowly progressive process of living to an arbitrary-defined old age, i.e., longevity. Whereas the genetic basis of rare premature aging syndromes has been elucidated, only a small fraction of the genetic determinants of longevity and life span, time from birth to death, have been identified. The latter point to the complexity of the process and involvement of myriad of genetic and non-genetic factors and hence, the diluted effect of each determinant on longevity. The genetic discoveries point to the involvement of the DNA damage and activation of the DNA damage response pathway, particularly in the premature aging syndromes. Likewise, the insulin/insulin-like growth factor 1/mTOR/FOXO pathways have emerged as major regulators of life span. A notable fraction of the genetic variants that are associated with life span is also associated with age-related cardiovascular diseases, such as coronary artery disease and dyslipidemia, which places cardiovascular aging at the core of human life span. The clinical impact of the discoveries pertains to the identification of the pathways that are involved in life span, which might serve as targets of interventions to prevent, slow, and even possibly reverse aging.

Identification of novel HLA-DQA1*03:02:03 allele using next-generation sequencing.

The HLA-DQA1*03:02:03 allele differs from HLA-DQA1*03:02:01:01 allele by a single nucleotide in codon 42.

Construction of a High-Density Genetic Map for Pitaya Using the Whole Genome Resequencing Approach

Pitaya (Hylocereus undatus) is one of the most economic fleshy fruit tree crops. This study aimed at producing a high-density linkage genetic map of pitaya based on the whole genome resequencing (WGrS) approach. For this purpose, a bi-parental F1 population of 198 individuals was generated and genotyped by WGrS. High-quality polymorphic 6434 single polymorphism nucleotide (SNP) markers were extracted and used to construct a high-density linkage map. A total of 11 linkage groups were resolved as expected in accordance with the chromosome number. The map length was 14,128.7 cM with an average SNP interval of 2.2 cM. Homology with the sequenced reference genome was described, and the physical and genetic maps were compared with collinearity analysis. This linkage map in addition to the available genomic resources will help for quantitative trait mapping, evolutionary studies and marker-assisted selection in the important Hylocereus species.

The FusX TALE Base Editor (FusXTBE) for Rapid Mitochondrial DNA Programming of Human Cells In Vitro and Zebrafish Disease Models In Vivo.

Functional analyses of mitochondria have been hampered by few effective approaches to manipulate mitochondrial DNA (mtDNA) and a lack of existing animal models. Recently a TALE-derived base editor was shown to induce C-to-T (or G-to-A) sequence changes in mtDNA. We report here the FusX TALE Base Editor (FusXTBE) to facilitate broad-based access to TALE mitochondrial base editing technology. TALE Writer is a de novo in silico design tool to map potential mtDNA base editing sites. FusXTBE was demonstrated to function with comparable activity to the initial base editor in human cells in vitro. Zebrafish embryos were used as a pioneering in vivo test system, with FusXTBE inducing 90+% editing efficiency in mtDNA loci as an example of near-complete induction of mtDNA heteroplasmy in vivo. Gene editing specificity as precise as a single nucleotide was observed for a protein-coding gene. Nondestructive genotyping enables single-animal mtDNA analyses for downstream biological functional genomic applications. FusXTBE is a new gene editing toolkit for exploring important questions in mitochondrial biology and genetics.

The Identification of Sialuria with Different Degrees of Intellectual Disabilities in Children and Adolescents

Background: Single nucleotide polymorphism/mutation in the R263L region of the allosteric site of the GNE gene produces a phenotype with an overproduction of intracellular levels of sialic acid and causes sialuria. In sialuria, a defective GNE gene, synthesized with lost feedback inhibition mechanism, produces many developmental delays and varying degrees of intellectual disabilities in children and adolescents. Several mutations in the epimerase and kinase domains exist that cause difficulty in getting a precise and exact effect of the GNE gene on the disease severity and sialic acid levels. This is the first study investigating the molecular basis of neuronal disorders exhibiting sialuria in Pakistani children/ adolescents.
 Methodology: The current study quantified the mRNA expression of the GNE gene and urinary sialic acid concentration by Realtime-qRT-PCR and Fluorimetric assays, respectively. The correlation between relative mRNA and urinary sialic acid levels was evaluated by using Pearson Bivariate correlations.
 Results: The data show that severely intellectually disabled (I.D.) patients showed significantly reduced mRNA expression levels of the GNE gene compared to controls. The concentrations of free sialic acid in urine were significantly reduced in severe I.D. patients compared to controls. Whereas patients with mild I.D. showed a two-fold increase in sialic acid levels when compared to controls. A significant correlation was found between an increased GNE mRNA and low urinary sialic acid levels from severe I.D. patients.
 Conclusion: The effect of the GNE gene is beyond hyposialylation that could hinder N-glycan structure and sialic acid biosynthesis. The study highlighted the possible involvement of sialic acid levels with different degrees of intellectual disabilities in Pakistani children and adolescents.

Mutation of the rice AN1-type zinc-finger protein gene ASL4 causes chloroplast development defects and seedling lethality.

Plant zinc-finger proteins play a crucial role in biosynthesis and plant development. However, it is not known whether certain zinc-finger proteins play a role in rice chloroplast development. In this study, a novel rice zinc-finger protein mutant asl4 (albino seedling lethality4), which exhibits an albino lethal phenotype at the seedling stage, was used. Chlorophyll fluorescence analysis and TEM were used to investigate features of the asl4 mutant. The genetic behaviour and function of ASL4 gene were then analysed thorough map-based cloning, transgenic complement and subcellular localization. The albino lethal phenotype was caused by a single nucleotide (G*) deletion mutation on the exon of the ASL4 (LOC_Os09g21710) gene. The ASL4 gene encoded a novel zinc-finger protein containing two ZnF-AN1 domains, which was localized to the nucleocytoplasm. The ASL4 transcripts were highly expressed in all leaves but relatively less in other tissues, suggesting its tissue-specific expression. The transcript levels of associated genes for Chl biosynthesis, photosynthesis and chloroplast development were severely suppressed in asl4 mutants. In conclusion, the absence of ASL4 function caused a defect in chloroplast development and seedling lethality. This is the first published report on the importance of the ZnF-AN1 type zinc-finger protein gene in chloroplast development in rice.

Identification of a novel HLA-DPB1*02:01:61 allele in an Indian individual.

The novel allele HLA-DPB1*02:01:61 as compared with HLA-DPB1*02:01:02 displays polymorphism by a single nucleotide at gDNA 9011 C->T.

NEW GENES, NEW TECHNIQUES IN NEUROMUSCULAR DISORDERS: O.12 Muscle biopsy and RNAseq in the diagnosis of titin related diseases

The ACMG/AMP guidelines for variant interpretation underline that splicing defects are an important mechanism of pathogenicity. In a standard diagnostic pipeline, in silico tools are needed to assess the possible effect on the splice of any identified genetic change (including synonymous variants) and a direct analysis of RNA, cDNA and protein is crucial to support variant classification. Due the sheer size of the TTN transcripts, the interpretation of titin variants is challenging and many unsolved myopathy patients (up to 6%) carry a single heterozygous TTN causative variant. We re-evaluated by SpliceAI all the possible single nucleotide changes in the titin coding sequence, identifying 9,732 variants predicted to alter the splicing (threshold 0.5). Of these, 2,078 are synonymous or missense variants and 317 are nonsense variants. Few previously reported causative ‘missense’ variants are predicted to impact the splicing. At the same time, we analysed by RNA sequencing 14 samples from patients with a confirmed recessive titinopathy, 24 samples from patients with a single heterozygous disease-causing variant in TTN and six from patients with a clinical suspicion of titinopathy. Variants causing a premature stop codon, located in exons out of the M-band, result in a nonsense mediated decay of titin transcripts. Vice versa, the in-depth characterization of 15 splice variants demonstrates that most of them cause in-frame losses or gains, still resulting in a near–full length protein. Using current bioinformatic approaches, RNAseq fails to identify elusive variants in most unsolved patients with heterozygous TTNtv and/or TTN-compatible phenotype, suggesting three possible scenarios: a causative role of the identified missense changes, more complex mechanisms involving heterozygous titin truncating variants, or alternative molecular diagnoses. At the same time, our study demonstrates the clinical importance of TTN transcript and protein studies in the diagnostic pipeline of neuromuscular disorders.

Sequence-based typing identification of the novel HLA-B*27:226 variant allele in a Chinese family.

HLA-B*27:226 differs from HLA-B*27:04:01 by a single nucleotide in exon 2 (91T→G, Y7D).

Impacts of single nucleotide deletions from the 3′ end of Senecavirus A 5′ untranslated region on activity of viral IRES and on rescue of recombinant virus

The 5′ untranslated region (UTR) of Senecavirus A (SVA) harbors an internal ribosome entry site (IRES), in which a pseudoknot structure is upstream of start codon AUG. Wild-type SVAs have a highly conserved 13-nt-sequence between the pseudoknot stem II (PKS-II)-forming motif and the AUG. In this study, a single nucleotide was deleted one by one from the 13-nt-sequence within a wild-type SVA minigenome. The result showed that neither mono- nor multi-nucleotide deletions abolished the IRES activity. Furthermore, a single nucleotide was deleted one by one from the 13-nt-sequence within a full-length SVA cDNA clone. The result indicated that nucleotide-deleting SVAs could be rescued from 1- to 5-nt-deleting cDNA clones, whereas only the 1- and 2-nt-deleting viruses were genetically stable during nine serial passages in vitro. Additionally, only the 1-nt-deleting SVA showed similar growth kinetics to that of the wild-type virus, suggesting that the pseudoknot-AUG distance was crucial for SVA replication.