Mutations In Sequence Research Articles

HGG-19. H3- AND IDH-WILDTYPE DIFFUSE PAEDIATRIC-TYPE HIGH-GRADE GLIOMA WITHMYCN-AMPLIFICATION: AN INDICATION CRITERION FOR LI-FRAUMENI SYNDROME TESTING

Abstract BACKGROUND: In the current World Health Organization (WHO) Classification of Tumors of the Central Nervous System (CNS) (5th edition), the introduction of a molecular layer to the integrated diagnosis not only helps to characterize and prognosticate paediatric CNS tumors but the molecular alteration may predict a potential therapeutic option with a targeted agent. Furthermore, certain gene alterations may be a presage of germline alterations or cancer predisposition syndromes, portending significant implications to the index patients and their families. We report a 5-year-old boy diagnosed with non-metastatic left hemispheric H3-wildtype and IDH-wildtype diffuse paediatric-type high-grade glioma with MYCN-amplification, who was subsequently found to have Li-Fraumeni syndrome. He presented with a 3-day history of facial asymmetry, unsteady gait and right sided weakness. There was no family history of malignancy. Initial MRI revealed a restricting and enhancing solid cystic lesion (5.9 x 4.3 x 5.8cm) in the left frontal-temporal-parietal region. Tumor debulking was performed. Histopathological examination by the local pathologist revealed features of high-grade glioma. Second pathology review with next generation sequencing confirmed H3-wildtype and IDH-wildtype diffuse paediatric-type high-grade glioma with MYCN-amplification and TP53 p.Y103* mutation. He was started on focal radiotherapy and oral temozolomide but succumbed to local and metastatic progressive disease. After genetic counselling, his DNA extracted from blood was sent for targeted exome sequencing and germline mutation of TP53 was demonstrated. Cascade screening of the family members were negative of TP53 mutation. Guerrini-Rousseau et al reported that 57% (n=7) of H3-wildtype and IDH-wildtype diffuse paediatric-type high-grade glioma with MYCN-amplification harboured germline TP53 pathogenic variants. This case further highlights the importance of screening of Li-Fraumeni syndrome in patient with H3-wildtype and IDH-wildtype diffuse paediatric-type high-grade glioma with MYCN-amplification even in the absence of a family history.

Hepatitis B Virus Genotypes and Subgenotypes Circulating in Infected Residents in a Country with High Vaccination Rate.

Despite the availability of a vaccine against hepatitis B virus (HBV), this infection still causes public health problems, particularly in susceptible populations. In Portugal, universal free vaccination started in 1994, and most HBV infections are diagnosed in immigrants from high-prevalence countries. Our aim was to assess the pattern of HBV genotypes/subgenotypes in samples collected between 2017 and 2021 from a convenience sample of 70 infected residents in Portugal. The HBV pol/HBsAg region was amplified and sequenced, allowing the analysis of RT sequences submitted to phylogenetic analysis and mutations assessment. A total of 37.1% of samples were from native Portuguese, aged 25-53 years (mean: 36.7 years), and the remaining samples were from individuals born outside of Portugal. A high diversity of HBV was identified: subgenotypes A1-A3 in 41.0% (16/39); D1, D3, and D4 in 30.7% (12/39); E in 23.1% (9/39); and F4 in 2.6% (1/39). Besides genotypes A and D, Portuguese were also infected with genotypes E and F, which are prevalent in Africa and South America, respectively. Resistance mutations in RT sequences were not found. The findings provide valuable insights for updating the HBV molecular epidemiology in Portugal. However, successful strategies to prevent and control the infection are still needed in the country, especially among susceptible and vulnerable populations.

A simple phylogenetic approach to analyze hypermutated HIV proviruses reveals insights into their dynamics and persistence during antiretroviral therapy.

Hypermutated proviruses, which arise in a single HIV replication cycle when host antiviral APOBEC3 proteins introduce extensive G-to-A mutations throughout the viral genome, persist in all people living with HIV receiving antiretroviral therapy (ART). But, the within-host evolutionary origins of hypermutated sequences are incompletely understood because phylogenetic inference algorithms, which assume that mutations gradually accumulate over generations, incorrectly reconstruct their ancestor-descendant relationships. Using > 1400 longitudinal single-genome-amplified HIV env-gp120 sequences isolated from six women over a median 18 years of follow-up - including plasma HIV RNA sequences collected over a median 9 years between seroconversion and ART initiation, and > 500 proviruses isolated over a median 9 years on ART - we evaluated three approaches for removing hypermutation from nucleotide alignments. Our goals were to 1) reconstruct accurate phylogenies that can be used for molecular dating and 2) phylogenetically infer the integration dates of hypermutated proviruses persisting during ART. Two of the tested approaches (stripping all positions containing putative APOBEC3 mutations from the alignment, or replacing individual putative APOBEC3 mutations in hypermutated sequences with the ambiguous base R) consistently normalized tree topologies, eliminated erroneous clustering of hypermutated proviruses, and brought env-intact and hypermutated proviruses into comparable ranges with respect to multiple tree-based metrics. Importantly, these corrected trees produced integration date estimates for env-intact proviruses that were highly concordant with those from benchmark trees that excluded hypermutated sequences, indicating that the corrected trees can be used for molecular dating. Use of these trees to infer the integration dates of hypermutated proviruses persisting during ART revealed that these spanned a wide age range, with the oldest ones dating to shortly after infection. This indicates that hypermutated proviruses, like other provirus types, begin to be seeded into the proviral pool immediately following infection, and can persist for decades. In two of the six participants, hypermutated proviruses differed from env-intact ones in terms of their age distributions, suggesting that different provirus types decay at heterogeneous rates in some hosts. These simple approaches to reconstruct hypermutated provirus' evolutionary histories, allow insights into their in vivo origins and longevity, towards a more comprehensive understanding of HIV persistence during ART.

Genomic analyses of withers height and linear conformation traits in German Warmblood horses using imputed sequence-level genotypes

BackgroundBody conformation, including withers height, is a major selection criterion in horse breeding and is associated with other important traits, such as health and performance. However, little is known about the genomic background of equine conformation. Therefore, the aim of this study was to use imputed sequence-level genotypes from up to 4891 German Warmblood horses to identify genomic regions associated with withers height and linear conformation traits. Furthermore, the traits were genetically characterised and putative causal variants for withers height were detected.ResultsA genome-wide association study (GWAS) for withers height confirmed the presence of a previously known quantitative trait locus (QTL) on Equus caballus (ECA) chromosome 3 close to the LCORL/NCAPG locus, which explained 16% of the phenotypic variance for withers height. An additional significant association signal was detected on ECA1. Further investigations of the region on ECA3 identified a few promising candidate causal variants for withers height, including a nonsense mutation in the coding sequence of the LCORL gene. The estimated heritability for withers height was 0.53 and ranged from 0 to 0.34 for the conformation traits. GWAS identified significantly associated variants for more than half of the investigated conformation traits, among which 13 showed a peak on ECA3 in the same region as withers height. Genetic parameter estimation revealed high genetic correlations between these traits and withers height for the QTL on ECA3.ConclusionsThe use of imputed sequence-level genotypes from a large study cohort led to the discovery of novel QTL associated with conformation traits in German Warmblood horses. The results indicate the high relevance of the QTL on ECA3 for various conformation traits, including withers height, and contribute to deciphering causal mutations for body size in horses.

Detecting Type and Index Mutation in Cancer DNA Sequence Based on Needleman–Wunsch Algorithm

Detecting DNA sequence mutations in cancer patients contributes to early identification and treatment of the disease, which ultimately enhances the effectiveness of treatment. Bioinformatics utilizes sequence alignment as a powerful tool for identifying mutations in DNA sequences. We used the Needleman-Wunsch algorithm to identify mutations in DNA sequence data from cancer patients. The cancer sequence dataset used includes breast, cervix uteri, lung, colon, liver and prostate cancer. Various types of mutations were identified, such as Single Nucleotide Variant (SNV)/substitution, insertion, and deletion, locate by the nucleotide index. The Needleman Wunch algorithm can detect type and index mutation with the average F1-scores 0.9507 for all types of mutations, 0.9919 for SNV, 0.7554 for insertion, and 0.8658 for deletion with a tolerance of 5 bp. The F1-scores obtained are not correlated with gene length. The time required ranges from 1.03 seconds for a 290 base pair gene to 3211.45 seconds for a gene with 16613 base pairs.

Advancing cancer MRD monitoring through tumor whole-genome informed, duplex ctDNA sequencing.

e15044 Background: Circulating tumor DNA (ctDNA), a cell-free DNA (cfDNA) present in the plasma of cancer patients, originates from cancer cells and serves as a crucial biomarker during cancer treatment. Despite its importance, the current limit of detection (LoD) of conventional ctDNA assays is suboptimal (0.01%; 10-4) for detecting ctDNA from microscopic residual and recurrent cancer tissues. To address this, we integrated two cutting-edge techniques: whole-genome sequencing (WGS) of primary cancer tissue (a mutation capture step) and duplex DNA sequencing-based cfDNA sequencing (mutation recapture steps). Methods: Thirteen colorectal cancer patients participated in this study. Primary tumor tissues and matched normal blood tissues were collected for WGS in the mutation capture step, while plasma samples for cfDNA sequencing in the mutation recapture steps were obtained just prior to colorectal cancer surgery and 1-month post-surgery follow-up. Tumor DNA and germline DNA were analyzed using CancerVision, a CLIA-certified clinical-grade cancer WGS platform, in the mutation capture step. In the mutation recapture steps, somatically acquired mutations from the previous step were tracked in cfDNA samples using the Concatenating Original Duplex for Error Correction (CODEC) technique, a duplex DNA sequencing technique that examines both Watson and Crick strands of double-stranded DNA molecules to discern true mutations from sequencing noise. Results: The median WGS depth of tumor and germline tissues was 40x and 20x, respectively. The mean depth of cfDNA WGS with CODEC was 25x, with a median duplex sequencing rate of 70%. Overall, 4,828-301,434 somatic base substitutions (SBSs) per sample were discovered in the capture step. Four of the thirteen tumors exhibited hypermutator characteristics with microsatellite instability features. The CODEC technique demonstrated excellent capability in tracing cancer-specific mutations in cfDNA sequencing with a technical sequencing error rate of 4 x 10-7, defining the maximum technical LoD, which is 250-fold lower errors than conventional sequencing. In our sample cohort, tumor fractions in cfDNA sequencing were robustly estimated from 0.001% (10-5), below the LoD of conventional MRD methods. Conclusions: Our integration of tumor-WGS-informed duplex cfDNA sequencing methods significantly enhances the sensitivity of sequencing-based MRD assays, achieving a technical LoD limit of 10-7 for detecting ctDNA in cancer patients. These approaches represent a breakthrough in monitoring MRD in real-world cancer patients.

Using a comprehensive atlas and predictive models to reveal the complexity and evolution of brain-active regulatory elements.

Most genetic variants associated with psychiatric disorders are located in noncoding regions of the genome. To investigate their functional implications, we integrate epigenetic data from the PsychENCODE Consortium and other published sources to construct a comprehensive atlas of candidate brain cis-regulatory elements. Using deep learning, we model these elements' sequence syntax and predict how binding sites for lineage-specific transcription factors contribute to cell type-specific gene regulation in various types of glia and neurons. The elements' evolutionary history suggests that new regulatory information in the brain emerges primarily via smaller sequence mutations within conserved mammalian elements rather than entirely new human- or primate-specific sequences. However, primate-specific candidate elements, particularly those active during fetal brain development and in excitatory neurons and astrocytes, are implicated in the heritability of brain-related human traits. Additionally, we introduce PsychSCREEN, a web-based platform offering interactive visualization of PsychENCODE-generated genetic and epigenetic data from diverse brain cell types in individuals with psychiatric disorders and healthy controls.

Does Salmonella diarizonae 58:r:z53 Isolated from a Mallard Duck Pose a Threat to Human Health?

Salmonella diarizonae (IIIb) is frequently isolated from reptiles and less frequently from birds and mammals. However, its isolation from invasive human infections has not been widely reported. Migratory mallard ducks are excellent bioindicators of pathogen presence and pathogen antibiotic resistance (AMR). We present the first isolation from a mallard duck in central Europe of the antibiotic-resistant Salmonella enterica subsp. diarizonae with the unique antigenic pattern 58:r:z53 and report its whole-genome sequencing, serosequencing, and genotyping, which enabled the prediction of its pathogenicity and comparison with phenotypic AMR. The isolated strain was highly similar to S. diarizonae isolated from humans and food. Twenty-four AMR genes were detected, including those encoding aminoglycoside, fluoroquinolone, macrolide, carbapenem, tetracycline, cephalosporin, nitroimidazole, peptide antibiotic, and disinfecting agent/antiseptic resistance. Six Salmonella pathogenicity islands were found (SPI-1, SPI-2, SPI-3, SPI-5, SPI-9, and SPI-13). An iron transport system was detected in SPI-1 centisome C63PI. Plasmid profile analyses showed three to be present. Sequence mutations in the invA and invF genes were noted, which truncated and elongated the proteins, respectively. The strain also harbored genes encoding type-III secretion-system effector proteins and many virulence factors found in S. diarizonae associated with human infections. This study aims to elucidate the AMR and virulence genes in S. enterica subsp. diarizonae that may most seriously threaten human health.

#2048 Exploring somatic mutation mechanisms in pre-cystic renal cells of autosomal dominant polycystic kidney disease patients

Abstract Background and Aims Genetic changes accumulate in our cells during a lifetime, at a rate that varies according to cell type and mutagen exposure [1]. Somatic mutations are the driving force of cancer and contribute to “second hit” disorders, such as Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is caused by inherited mutations that disrupt one allele of PKD1 or PKD2 genes. Somatic loss of the second allele triggers tubule cell clonal expansion and the formation of renal cysts. Given the high number of cysts that form during a lifetime, somatic mutation rates may be abnormally high in the kidney of ADPKD patients. The identification of factors enhancing somatic mutation rates can lead to preventive strategies to limit cyst formation. Metabolic alterations, i.e. increased glutamine utilization and reduced urea cycle, have been shown to induce somatic mutations in cancer [2]. Loss of urea cycle increases pyrimidine synthesis, which unbalances the nucleotide pools and results in a distinct signature of single base substitutions (SBSs) [1]. Similar metabolic changes have been observed in ADPKD [3, 4]. Thus, we sought to test metabolism-driven somatic mutagenesis in pre-cystic kidneys and its contribution to second hit mutations in ADPKD. Method We clonally expanded normal kidney cells from human urine samples and performed a gene expression study by qPCR. We studied 31 clones from 4 ADPKD patients (age range: 25-45) with truncating mutations in PKD1 and normal kidney function, despite a clear cystic phenotype. ADPKD cells were compared to 55 clones from 5 healthy volunteers (age range: 24-53), and 32 clones from 6 patients with another cystic kidney disease, Von Hippel Lindau disease (VHL; age range: 29-56). A subset of clones (n = 19 controls, n = 12 ADPKD) was subjected to whole genome sequencing and somatic mutation analysis. Results Urines of ADPKD patients contained higher numbers of cells that expanded in vitro, compared to both control (p = 0.0008) and VHL individuals (p = 0.01). Gene expression analyses showed that irrespective of genetic background, all cultured clones originated from the kidney tubule epithelium (high PAX2 and PAX8), and some originated from damaged tubules (VCAM1, KIM1). We tested the hypothesized metabolic rewiring by analyzing expression levels of enzymes involved in glutamine utilization, urea cycle, and pyrimidine biosynthesis. Except for ASNS (Asparagine Synthetase), which was higher in ADPKD vs control clones (p = 0.0098), no gene-expression differences were observed. However, clones from ADPKD patients showed signs of the metabolic rewiring responsible for increased pyrimidine production, i.e. a positive correlation (r = 0.691; p < 0.0001) between urea cycle enzymes downregulation and upregulation of the pyrimidine synthesis enzyme CAD. Since excessive pyrimidines lead to mutation [2], we analyzed the number of somatic SBSs per genome, after filtering for germline variants. We did not find increased mutation rates in ADPKD compared to controls, but an analogous, linear increase of mutations with age and similar levels of the pyrimidine-rich mutational signature. Conclusion Urine-derived kidney tubule epithelial cells with heterozygous truncating mutations in PKD1 exhibit certain characteristics of metabolic reprogramming typical of kidneys from ADPKD patients with advanced pathology. Nevertheless, in the limited number of pre-cystic cells that we have analyzed, this metabolic reprogramming did not translate into an excess of somatic mutations.

Rare genetic disorders that impair parathyroid hormone synthesis, secretion, or bioactivity provide insights into the diagnostic utility of different parathyroid hormone assays.

Parathyroid hormone (PTH) is the major peptide hormone regulator of blood calcium homeostasis. Abnormal PTH levels can be observed in patients with various congenital and acquired disorders, including chronic kidney disease (CKD). This review will focus on rare human diseases caused by PTH mutations that have provided insights into the regulation of PTH synthesis and secretion as well as the diagnostic utility of different PTH assays. Over the past years, numerous diseases affecting calcium and phosphate homeostasis have been defined at the molecular level that are responsible for reduced or increased serum PTH levels. The underlying genetic mutations impair parathyroid gland development, involve the PTH gene itself, or alter function of the calcium-sensing receptor (CaSR) or its downstream signaling partners that contribute to regulation of PTH synthesis or secretion. Mutations in the pre sequence of the mature PTH peptide can, for instance, impair hormone synthesis or intracellular processing, while amino acid substitutions affecting the secreted PTH(1-84) impair PTH receptor (PTH1R) activation, or cause defective cleavage of the pro-sequence and thus secretion of a pro- PTH with much reduced biological activity. Mutations affecting the secreted hormone can alter detection by different PTH assays, thus requiring detailed knowledge of the utilized diagnostic test. Rare diseases affecting PTH synthesis and secretion have offered helpful insights into parathyroid biology and the diagnostic utility of commonly used PTH assays, which may have implications for the interpretation of PTH measurements in more common disorders such as CKD.

The futuristic applications of transition metal dichalcogenides for cancer therapy.

The second-most common cause of death resulting from genetic mutations in DNA sequences is cancer. The difficulty in the field of anticancer research is the application of the traditional methods, which also affects normal cells. Mutations, genetic replication alterations, and chromosomal abnormalities have a direct impact on the effectiveness of anticancer drugs at different stages. Presently, therapeutic techniques utilize nanotechnology, transition metal dichalcogenides (TMDCs), and robotics. TMDCs are being increasingly employed in tumor therapy and biosensing applications due to their biocompatibility, adjustable bandgap, versatile functionality, exceptional photoelectric properties, and wide range of applications. This study reports the advancement of nanoplatforms based on TMDCs that are specifically engineered for responsive and intelligent cancer therapy. This article offers a thorough examination of the current challenges, future possibilities for theranostic applications using TMDCs, and recent progress in employing TMDCs for cancer therapy. Currently, there is significant interest in two-dimensional (2D) TMDCs nanomaterials as ultrathin unique physicochemical properties. These materials have attracted attention in various fields, including biomedicine. Due to their inherent ability to absorb near-infrared light and their exceptionally large surface area, significant efforts are being made to prepare multifunctional nanoplatforms based on 2D TMDCs.

Prime editing-mediated correction of the leptin receptor in muscle cells of db/db mice.

Genetic diseases can be caused by monogenic diseases, which result from a single gene mutation in the DNA sequence. Many innovative approaches have been developed to cure monogenic genetic diseases, namely by genome editing. A specific type of genomic editing, prime editing, has the potential advantage to edit the human genome without requiring double-strand breaks or donor DNA templates for editing. Additionally, prime editing does not require a precisely positioned protospacer adjacent motif (PAM) sequence, which offers flexible target and more precise genomic editing. Here we detail a novel construction of a prime editing extended guide RNA (pegRNA) to target mutated leptin receptors in B6.BKS(D)-Leprdb/J mice (db/db mice). The pegRNA was then injected into the flexor digitorum brevis (FDB) muscle of db/db mice to demonstrate in vivo efficacy, which resulted in pegRNA mediated base transversion at endogenous base transversion. Genomic DNA sequencing confirmed that prime editing could correct the mutation of leptin receptor gene in db/db mice. Furthermore, prime editing treated skeletal muscle exhibited enhanced leptin receptor signals. Thus, the current study showed in vivo efficacy of prime editing to correct mutant protein and rescue the physiology associated with functional protein.

Defining a Haplotype Encompassing the LCORL-NCAPG Locus Associated with Increased Lean Growth in Beef Cattle.

Numerous studies have shown genetic variation at the LCORL-NCAPG locus is strongly associated with growth traits in beef cattle. However, a causative molecular variant has yet to be identified. To define all possible candidate variants, 34 Charolais-sired calves were whole-genome sequenced, including 17 homozygous for a long-range haplotype associated with increased growth (QQ) and 17 homozygous for potential ancestral haplotypes for this region (qq). The Q haplotype was refined to an 814 kb region between chr6:37,199,897-38,014,080 and contained 218 variants not found in qq individuals. These variants include an insertion in an intron of NCAPG, a previously documented mutation in NCAPG (rs109570900), two coding sequence mutations in LCORL (rs109696064 and rs384548488), and 15 variants located within ATAC peaks that were predicted to affect transcription factor binding. Notably, rs384548488 is a frameshift variant likely resulting in loss of function for long isoforms of LCORL. To test the association of the coding sequence variants of LCORL with phenotype, 405 cattle from five populations were genotyped. The two variants were in complete linkage disequilibrium. Statistical analysis of the three populations that contained QQ animals revealed significant (p < 0.05) associations with genotype and birth weight, live weight, carcass weight, hip height, and average daily gain. These findings affirm the link between this locus and growth in beef cattle and describe DNA variants that define the haplotype. However, further studies will be required to define the true causative mutation.

Discrimination of Genetic Biomarkers of Disease through Machine-Learning-Based Hypothesis Testing of Direct SERS Spectra of DNA and RNA.

Cancer is globally a leading cause of death that would benefit from diagnostic approaches detecting it in its early stages. However, despite much research and investment, cancer early diagnosis is still underdeveloped. Owing to its high sensitivity, surface-enhanced Raman spectroscopy (SERS)-based detection of biomarkers has attracted growing interest in this area. Oligonucleotides are an important type of genetic biomarkers as their alterations can be linked to the disease prior to symptom onset. We propose a machine-learning (ML)-enabled framework to analyze complex direct SERS spectra of short, single-stranded DNA and RNA targets to identify relevant mutations occurring in genetic biomarkers, which are key disease indicators. First, by employing ad hoc-synthesized colloidal silver nanoparticles as SERS substrates, we analyze single-base mutations in ssDNA and RNA sequences using a direct SERS-sensing approach. Then, an ML-based hypothesis test is proposed to identify these changes and differentiate the mutated sequences from the corresponding native ones. Rooted in "functional data analysis," this ML approach fully leverages the rich information and dependencies within SERS spectral data for improved modeling and detection capability. Tested on a large set of DNA and RNA SERS data, including from miR-21 (a known cancer miRNA biomarker), our approach is shown to accurately differentiate SERS spectra obtained from different oligonucleotides, outperforming various data-driven methods across several performance metrics, including accuracy, sensitivity, specificity, and F1-scores. Hence, this work represents a step forward in the development of the combined use of SERS and ML as effective methods for disease diagnosis with real applicability in the clinic.

Conservation of a Chromosome 8 Inversion and Exon Mutations Confirm Common Gulonolactone Oxidase Gene Evolution Among Primates, Including H. Neanderthalensis

Ascorbic acid functions as an antioxidant and facilitates other biochemical processes such as collagen triple helix formation, and iron uptake by cells. Animals which endogenously produce ascorbic acid have a functional gulonolactone oxidase gene (GULO); however, humans have a GULO pseudogene (GULOP) and depend on dietary ascorbic acid. In this study, the conservation of GULOP sequences in the primate haplorhini suborder were investigated and compared to the GULO sequences belonging to the primates strepsirrhini suborder. Phylogenetic analysis suggested that the conserved GULOP exons in the haplorhini primates experienced a high rate of mutations following the haplorhini/strepsirrhini divergence. This high mutation rate has decreased during the evolution of the haplorhini primates. Additionally, indels of the haplorhini GULOP sequences were conserved across the suborder. A separate analysis for GULO sequences and well-conserved GULOP sequences focusing on placental mammals identified an in-frame GULO sequence in the Brazilian guinea pig, and a potential GULOP sequence in the pika. Similar to haplorhini primates, the guinea pig and lagomorph species have experienced a high substitution rate when compared to the mammals used in this study. A shared synteny to examine the conservation of local genes near GULO/GULOP identified a conserved inversion around the GULO/GULOP locus between the haplorhini and strepsirrhini primates. Fischer’s exact test did not support an association between GULOP and the chromosomal inversion. Mauve alignment showed that the inversion of the length of the syntenic block that the GULO/GULOP genes belonged to was variable. However, there were frequent rearrangements around ~ 2 million base pairs adjacent to GULOP involving the KIF13B and MSRA genes. These data may suggest that genes acquiring deleterious mutations in the coding sequence may respond to these deleterious mutations with rapid substitution rates.

Phylogenetic Analysis and Mutation of Sars-Cov-2 in Bats in Karst Malang City, Indonesia

Introduction: A group of people in China were hospitalized with an initial diagnosis of pneumonia of unknown cause. The patients were linked to a wholesale wet seafood and animal market in Wuhan, Hubei Province, China. The disease has spread to other provinces in China, Thailand, Japan, and South Korea in less than a month. SARS-CoV-2 was found to originate from bats. Therefore, this research aims to analyze SARS-CoV-2 mutation in bats in Malang Karst, Indonesia. Methods: Other bat body parts used as research samples include the brain, liver, kidneys, intestines, pancreas, fetus, blood, lungs, and ectoparasites. The samples were taken separately and placed in a container containing 10% PBF. For further analysis, we used RNA Extraction, Real-Time PCR, Sequencing, and CoV Gisaid mutation analysis software to analyze the sequencing data. Then, EMBL software will be used to analyze the phylogenetically. Results and Discussion: There was 1 sample that showed a positive result for Covid-19, namely the intestine of the Cynoptera brachyotis species. There were differences between SARS-CoV-2 in bats in Malang Karst in Indonesia compared to SARS-CoV from 2000 to 2019. The spike protein's receptor binding domain (RBD) is the most variable part of the coronavirus genome. Conclusion: From the research results, one positive sample was obtained using Real-Time PCR, and based on mutation analysis, mutations were found in SARS-CoV-2 against the SARS-CoV virus from 2000-2019. Further research is needed, especially regarding SARS-CoV-2 as a vaccine.

In vitro data suggest a role for PMS2 Kozak sequence mutations in Lynch syndrome risk

Lynch syndrome (LS) is the most common hereditary cancer syndrome. Heterozygous loss-of-function variants in PMS2 are linked to LS. While these variants are not directly cancer causing, reduced PMS2 function results in the accumulation of somatic variants and increased cancer risk over time due to DNA mismatch repair dysfunction. It is reasonable that other types of genetic variation that impact the expression of PMS2 may also contribute to cancer risk. The Kozak sequence is a highly conserved translation initiation motif among higher eukaryotes and is defined as the nine base pairs upstream of the translation start codon through the first four bases of the translated sequence (5'-[GTT]GCATCCATGG-3'; human PMS2: NM_000535.7). While Kozak sequence variants in PMS2 have been reported in ClinVar in patients with suspected hereditary cancer, all variants upstream of the translation start site are currently classified as variants of undetermined significance (VUSs). We hypothesized that variants significantly disrupting the Kozak sequence of PMS2 would decrease PMS2 protein expression, contributing to increased cancer risk over time. Using a dual-luciferase reporter plasmid and site-directed mutagenesis, we generated the wild-type human PMS2 and the ClinVar VUSs within the PMS2 Kozak sequence. Besides the c.1A>C variant, which is already known to be pathogenic, we implicate six additional variants as American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) pathogenic supporting (PP) variants and classify ten as benign supporting (BP). In summary, we present a method developed for the classification of human PMS2 Kozak sequence variants that can contribute to the re-classification of VUSs identified in patients.

Polysaccharide intercellular adhesin and proper phospholipid composition are important for aggregation in Tetragenococcus halophilus SL10.

Aggregating strains of Tetragenococcus halophilus tend to be trapped during soy sauce mash-pressing process and are, therefore, critical for clear soy sauce production. However, the precise molecular mechanism involved in T. halophilus aggregation remains elusive. In previous studies, we isolated a number of aggregating strains, including T. halophilus AB4 and AL1, and showed that a cell surface proteinaceous aggregation factor is responsible for their aggregation phenotype. In the present study, we explored the role of polysaccharide intercellular adhesin (PIA) in aggregate formation in T. halophilus SL10, isolated from soy sauce. SL10 exhibited similar aggregation to AB4 and AL1 but formed a non-uniform precipitate with distinctive wrinkles at the bottom of the test tube, unlike AB4 and AL1. Insertion sequence mutations in each gene of the ica operon diminished aggregation and PIA production, highlighting the critical role of IcaADBC-mediated PIA production in T. halophilus aggregation. Furthermore, two non-aggregating cardiolipin synthase (cls) gene mutants with intact ica operon did not produce detectable PIA. Phospholipid composition analysis in cls mutants revealed a decrease in cardiolipin and an increase in phosphatidylglycerol levels, highlighting the association between phospholipid composition and PIA production. These findings provide evidence for the pivotal role of cls in PIA-mediated aggregation and lay the foundation for future studies to understand the intricate networks of the multiple aggregation factors governing microbial aggregation.IMPORTANCEAggregation, commonly observed in various microbes, triggers biofilm formation in pathogenic variants and plays a beneficial role in efficient food production in those used for food production. Here, we showed that Tetragenococcus halophilus, a microorganism used in soy sauce fermentation, forms aggregates in a polysaccharide intercellular adhesin (PIA)-mediated manner. Additionally, we unveiled the relationship between phospholipid composition and PIA production. This study provides evidence for the presence of aggregation factors in T. halophilus other than the proteinaceous aggregation factor and suggests that further understanding of the coordinated action of these factors may improve clarified soy sauce production.

Evolutionary Changes in Primate Glutamate Dehydrogenases 1 and 2 Influence the Protein Regulation by Ligands, Targeting and Posttranslational Modifications.

There are two paralogs of glutamate dehydrogenase (GDH) in humans encoded by the GLUD1 and GLUD2 genes as a result of a recent retroposition during the evolution of primates. The two human GDHs possess significantly different regulation by allosteric ligands, which is not fully characterized at the structural level. Recent advances in identification of the GDH ligand binding sites provide a deeper perspective on the significance of the accumulated substitutions within the two GDH paralogs. In this review, we describe the evolution of GLUD1 and GLUD2 after the duplication event in primates using the accumulated sequencing and structural data. A new gibbon GLUD2 sequence questions the indispensability of ancestral R496S and G509A mutations for GLUD2 irresponsiveness to GTP, providing an alternative with potentially similar regulatory features. The data of both GLUD1 and GLUD2 evolution not only confirm substitutions enhancing GLUD2 mitochondrial targeting, but also reveal a conserved mutation in ape GLUD1 mitochondrial targeting sequence that likely reduces its transport to mitochondria. Moreover, the information of GDH interactors, posttranslational modification and subcellular localization are provided for better understanding of the GDH mutations. Medically significant point mutations causing deregulation of GDH are considered from the structural and regulatory point of view.

An enzyme-free and ratiometric biosensor for single nucleotide polymorphism with portable electrochemical system

Portable, low-cost and efficient biosensing systems are valuable for single nucleotide polymorphisms (SNPs) analysis, particularly in resource-limited fields. Herein, a rapid, enzyme-free and ratiometric SNPs biosensor with portable electrochemical system enabling simultaneous recognition and signal amplification is proposed. To demonstrate its versatility and effectiveness, we have selected the human cancer-related KRAS gene and the soybean mosaic virus resistance Rsv3 gene as our analysis models. Our biosensor is constructed based on a combination of strand displacement reactions and hybridization chain reactions (HCR). This unique design enables to differentiate between mutant (MT) and wild (WD) sequences based on the differential release of HCR products on the electrode surfaces. Importantly, this enzyme-free SNPs biosensor concurrently achieves both SNPs recognition and signal amplification, significantly reducing the reaction time to just 40 minutes. Furthermore, we have developed a miniaturized biosensor system that incorporates screen printed gold electrodes (SPGE) and smartphones. This system is integrated with a ratiometric strategy with an limit of detection as low as 1.2 fM, enhancing accuracy and eliminating external interference. This simple SNPs biosensing system possesses high reliability, positioning it as a versatile biosensor for the determination of various SNPs mutations.