Non-synonymous Mutations Research Articles

Genetic Variants Affecting FADS2 Enzyme Dynamics and Gene Expression in Cogenetic Oysters with Different PUFA Levels Provide New Tools to Improve Unsaturated Fatty Acids

Long-chain polyunsaturated fatty acids (LC-PUFAs) are crucial for human health and cannot be produced internally. Bivalves, such as oysters, serve as valuable sources of high-quality PUFAs. The enzyme fatty acid desaturase (FADS) plays a key role in the metabolism of LC-PUFAs. In this study, we conducted a thorough genome-wide analysis of the genes belong to the FADS family in Crassostrea gigas and Crassostrea angulata, with the objective of elucidating the function of the FADS2 and investigating the genetic variations that affect PUFA biosynthesis. We identified six FADS genes distributed across four chromosomes, categorized into three subfamilies. The coding region of FADS2 revealed five non-synonymous mutations that were shown to influence protein structure and stability through molecular dynamics simulations. The promoter region of FADS2 contains ten SNPs and three indels significantly correlated with PUFA content. These genetic variations may explain the differences in PUFA levels observed between the two oyster species and could have potential applications in enhancing PUFA content. This study improves the molecular understanding of PUFA metabolism in oysters and presents a potential strategy for selecting oysters with high PUFA levels.

The F204S mutation in adrenodoxin oxidoreductase drives salinomycin resistance in Eimeria tenella

Salinomycin is a polyether ionophore widely used for the treatment of coccidiosis in poultry. However, the emergence of coccidia strains resistant to salinomycin presents challenges for control efforts, and the mechanisms underlying this resistance in Eimeria remain inadequately understood. In this study, 78 stable salinomycin-resistant strains were generated through experimental evolution approaches. Whole-genome sequencing of salinomycin-resistant Eimeria tenella isolates revealed single nucleotide polymorphisms (SNPs), with 12 candidate genes harboring nonsynonymous mutations identified. To confirm the candidate gene responsible for conferring salinomycin resistance, we leveraged reverse genetic strategies and identified a key amino acid substitution (F204S) in adrenodoxin oxidoreductase (EtADR), which markedly reduced susceptibility to salinomycin. Our results elucidate the complex interactions among salinomycin resistance, parasite fitness, point mutations, and the structure of EtADR, laying the foundation for future studies on drug resistance in Eimeria and contributing to the development of targeted control strategies.

Cancer-type somatic mutations in saccular cerebral aneurysms.

Intracranial aneurysms (IAs) are a major cause of subarachnoidal hemorrhage (SAH) which can have a significant morbidity and mortality. The processes underlying the aneurysm development remains unclear. We performed whole exome sequencing of DNA derived from 20 saccular cerebral aneurysms of 20 patients, followed by somatic variant calling. Eleven (55%) of the 20 patients had detectable nonsynonymous somatic mutations and in total, 48 mutations were detected in the aneurysm samples. The mutations were highly enriched in cancer-related genes and 77% were predictably deleterious. A p.Tyr562Asp somatic mutation was detected in the PDGFRB gene; somatic mutations at the same codon have been reported in fusiform cerebral aneurysms. These results widen the concept on the role of somatic mutations in cerebral aneurysms, indicating their possible role in the more common saccular aneurysm, similarly to the rarer fusiform aneurysm.

Transethnic analysis identifies SORL1 variants and haplotypes protective against Alzheimer's disease.

The SORL1 locus exhibits protective effects against Alzheimer's disease (AD) across ancestries, yet systematic studies in diverse populations are sparse. Logistic regression identified AD-associated SORL1 haplotypes in East Asian (N=5249) and European (N=8588) populations. Association analysis between SORL1 haplotypes and AD-associated traits or plasma biomarkers was conducted. The effects of non-synonymous mutations were assessed in cell-based systems. Protective SORL1 variants/haplotypes were identified in the East Asian and European populations. Haplotype Hap_A showed a strong protective effect against AD in East Asians, linked to less severe AD phenotypes, higher SORL1 transcript levels, and plasma proteomic changes. A missense variant within Hap_A, rs2282647-C allele, was linked to a lower risk of AD and decreased expression of a truncated SORL1 protein isoform. Our transethnic analysis revealed key SORL1 haplotypes that exert protective effects against AD, suggesting mechanisms of the protective role of SORL1 in AD. We examined the AD-protective mechanisms of SORL1 in the general population across diverse ancestral backgrounds by jointly analyzing data from three East Asian cohorts (ie, mainland China, Hong Kong, and Japan) and a European cohort. Comparative analysis unveiled key ethnic-specific SORL1 genetic variants and haplotypes. Among these, the SORL1 minor haplotype, Hap_A, emerged as the primary AD-protective factor in East Asians. Hap_A exerts significant AD-protective effects in both APOE ε4 carriers and non-carriers. SORL1 haplotype Hap_A is associated with cognitive function, brain volume, and the activity of specific neuronal and immune-related pathways closely connected to AD risk. Protective variants within Hap_A are linked to increased SORL1 expression in human tissues. We identified an isoform-specific missense variant in Hap_A that modifies the function and levels of a truncated SORL1 protein isoform that is poorly investigated.

Detection of Genetic Variations in Children with Tetralogy of Fallot Using Whole Exome Sequencing Technology Integrated Bioinformatics Analysis.

Background: Tetralogy of Fallot (TOF) is the most common cyanotic heart defect in newborns, with a complex etiology and genetic variation considered to be one of the main pathogenic factors. Identifying genetic variations associated with TOF has important clinical value for understanding its pathogenesis, patient susceptibility, and prognosis of patients with TOF. Therefore, this study aimed to identify potential pathogenic genes of TOF through comprehensive genetic analysis. Materials and Methods: In this study, we employed whole exome sequencing (WES) of the DNA of 47 Chinese children who received surgical TOF treatment at the Children's Hospital of Zhejiang University of Medicine and processed for DNA extraction and quantification of the DNA following WES using the Illumina NovaSeq platform. WES data undergo strict quality control and analysis processes including alignment, postprocessing, variant calling, annotation, and prioritization. Key tools, such as GATK's haplotype calling module and Annotate Variation, were used for variant annotation. In addition, by combining bioinformatics tools such as SIFT, Polyphen2, and Clin Pred, we evaluated the potential impact of nonsynonymous mutations on protein function and referred to relevant literature to support our prediction. Results: Comprehensive data analysis and quality assessment analysis corroborated the data generated from the WES dataset of 47 patients with TOF. Interpreting variants from the perspective of clinical pathogenicity results revealed a novel polymorphism and variant associated with TOF. The identified genetic results revealed evidence for a major contribution of MUTYH, RARB, GFM1, PDZD2, CEP57, DCPS, POMT2, BUB1B, CYP19A1, MAZ, USP10, and TCF3 and provided novel findings for functionally interacting proteins associated with the pathomechanism of TOF. Seven pathogenic variants related to TOF were detected, most of which were previously unreported in this cohort. Conclusions: The genetic variations discovered in this study emphasize the importance of genetic factors in the pathogenesis of TOF, revealing its complex molecular pathways and protein-protein interactions. The study of genetic diversity provides a new perspective for understanding the etiology of TOF and promotes an in-depth exploration of its pathological mechanisms. These findings lay the foundation for subsequent clinical research and the development of treatment strategies.

Repeat-induced point mutations driving Parastagonospora nodorum genomic diversity are balanced by selection against non-synonymous mutations

Parastagonospora nodorum is necrotrophic fungal pathogen of wheat with significant genomic resources. Population-level pangenome data for 173 isolates, of which 156 were from Western Australia (WA) and 17 were international, were examined for overall genomic diversity and effector gene content. A heterothallic core population occurred across all regions of WA, with asexually-reproducing clonal clusters in dryer northern regions. High potential for SNP diversity in the form of repeat-induced point mutation (RIP)-like transitions, was observed across the genome, suggesting widespread ‘RIP-leakage’ from transposon-rich repetitive sequences into non-repetitive regions. The strong potential for RIP-like mutations was balanced by negative selection against non-synonymous SNPs, that was observed within protein-coding regions. Protein isoform profiles of known effector loci (SnToxA, SnTox1, SnTox3, SnTox267, and SnTox5) indicated low-levels of non-synonymous and high-levels of silent RIP-like mutations. Effector predictions identified 186 candidate secreted predicted effector proteins (CSEPs), 69 of which had functional annotations and included confirmed effectors. Pangenome-based effector isoform profiles across WA were distinct from global isolates and were conserved relative to population structure, and may enable new approaches for monitoring crop disease pathotypes.

The spread of molecular markers of artemisinin partial resistance and diagnostic evasion in Eritrea: a retrospective molecular epidemiology study

Eritrea was the first African country to discontinue the use of histidine rich protein 2 (HRP2)-detecting rapid diagnostic tests (RDTs) for malaria diagnosis following reports of a high prevalence of pfhrp2/3-deleted Plasmodium falciparum parasites causing false-negative results in the country. Eritrea was also the first African country to report partial artemisinin resistance due to the Pfalciparum kelch13 (pfk13) Arg622Ile mutation. We aimed tocharacterise the spatial distribution of pfk13 mutants and their interactions with pfhrp2/3 deletions in Eritrea andto assess the role of the use of HRP2-detecting RDTs and antimalarial (artesunate-amodiaquine) therapy in the spread of the two variants. We conducted a retrospective molecular epidemiological analysis of pfk13 mutations and pfhrp2/3 deletions in existing Pfalciparum-infected blood samples collected as part of previous pfhrp2/3 deletion and severe malaria studies. Samples were collected in March, 2016and between September, 2018, and January, 2020, from symptomatic patients seeking care at 15health centres in four administration zones (Semenawi Keyih Bahri, Gash Barka, Anseba, and Debub) in Eritrea. Afragment spanning the propeller region of pfk13 was amplified from samples and sequenced using Sanger sequencing or targeted amplicon sequencing to identify genetic mutations. Deletions of pfhrp2/3 genes in samples were determined using multiplex quantitative PCR. Parasite haplotypes and genetic relatedness of parasite haplotypes were determined previously using microsatellite marker typing. The primary objective was to determine the prevalence of pfk13 mutations at health centres and administrative zones. The secondary objective was to investigate whether pfk13 mutants and pfhrp2/3 deleted parasites converge. We sequenced 50samples collected in March, 2016from the Semenawi Keyih Bahri zone and identified no pfk13 mutations. By contrast, in 587samples included in this study that were collected from health centres in GashBarka, Anseba, and Debub in 2018-20, we detected five different single non-synonymous mutations: Glu605Lys, Arg622Ile, Asn657Lys, Lys658Glu, and Ser679Leu. The most prevalent mutation was pfk13 Arg622Ile, which was detected in samples collected from all nine health centres where more than five samples were available across all three administration zones, with an overall prevalence of 11·9% (70 of 587samples; range 5·9-28·0%). Weidentified 22unique pfk13 Arg622Ile mutant haplotypes among 26samples tested, of which 13 (59·1%) were genetically related, whereas the remaining nine (40·9%) were not. The prevalence of pfk13 Arg622Ile was significantly higher in parasites with a single pfhrp3 deletion (46 [18·0%] of 255samples) than in parasites withoutpfhrp2/3 deletions (ten [6·2%] of 161samples; odds ratio 3·89 [95% CI 1·59-7·61]; p=0·0006) and with dual pfhrp2/3-deleted parasites (13 [9·0%] of 145; 2·23 [1·13-4·68]; p=0·018). The geographical spread of the pfk13 Arg622Ile mutation might have initially resulted from the clonal expansion and spread of pfhrp2/3 deletions under the test-and-treat policy using HRP2-detecting RDTs. Subsequently, selective pressure from artemisinin combination therapy could have further facilitated the spread of both pfk13 Arg622Ile and pfhrp2/3 deletions. Continuous monitoring of trends in pfk13 and pfhrp2/3 variants is needed to inform effective malaria control and elimination strategies in Eritrea and other African countries. US Department of Defense Armed Forces Health Surveillance Division, Global Emerging Infections Surveillance Branch (AFHSD/GEIS), and Wellcome Trust.

Molecular analysis of the 2022 mpox outbreak and antiviral activity of dihydroorotate dehydrogenase inhibitors against orthopoxviruses

Monkeypox virus (MPXV) has caused a large pandemic outbreak in 2022 with more than 90.000 confirmed cases and 181 deaths. Notably, signs of microevolution and host adaption have been observed. Here, we demonstrate that viral genomes from Franconia, Bavaria acquired different mutations. Three isolates obtained from diagnostic samples, submitted from suspected Mpox cases, show differences in their replication capacities. One MPXV isolate which shows the fastest replication kinetics and higher viral loads, possesses a unique non-synonymous mutation (D616L) in the A11L protein (gene OPG136), which encodes for a protein that is part of a major viral core structure. In regard to pandemic preparedness and future outbreaks, we analyzed the antiviral activity of dihydroorotate dehydrogenase (DHODH) inhibitors, and show that they are active against MPXV, vaccinia virus (VACV), and cowpox virus (CPXV) and therefore likely against orthopoxviruses in general. In agreement with that, we also demonstrated that chemical optimization leads to compounds with EC50 values in the sub-nanomolar range, associated with low cytotoxicity, which forms a good basis for future drug development from this chemical series.

Detection and Molecular Characterization of Avian Encephalomyelitis Virus in India: A Preliminary Report

Tremovirus A, previously recognized as Avian encephalomyelitis virus (AEV) is, one of the viruses that have been reported to be associated with nervous and enteric disorders in young birds. In Kerala, there has been an increase in the cases of nervous and enteric diseases in chickens. The detection and characterization of other viruses associated with nervous and enteric viruses in chickens has been established in Kerala. While there have been reports of nervous and enteric diseases in chickens in the region, no previous research had focused on AEV as the causative agent. A total of 50 samples were tested to detect AEV by reverse-transcriptase polymerase chain reaction (RT-PCR) targeting the 5' untranslated region (5'UTR) of AEV. Of the samples tested, one (2%) was found to be positive for the virus. The most immunogenic protein of AEV is VP1 protein forming the major capsid protein. The VP1gene segment of the detected virus isolate was amplified and molecular characterization was done. The phylogenetic analysis of the VP1 gene sequence revealed that the virus was identical to AEV viruses detected in Hungary, China, and Hong Kong (64% similarity). The current isolate showed a nonsynonymous mutation of I184V which was noticed only in Hungary isolate. The results of the study indicate that AEV is rarely present in chickens of Kerala, India, and that its occurrence is very low. Since the virus is capable of inducing nervous signs, mortality, and economic losses, measures are to be taken to control the spread of the virus in chickens in Kerala, India.

Comparison of dN/dS ratios shows little evidence for faster-Z effect in Furcifer chameleons after controlling for gene-specific evolutionary rates.

The faster-X/Z effect hypothesis states that genes linked to X/Z chromosomes should accumulate mutations faster than autosomal genes. Although faster evolution of X/Z-linked genes has been reported in several plant and animal lineages, conflicting results have been reported in others. We examined the faster-Z effect in chameleons of the genus Furcifer, a lineage with differentiated ZZ/ZW chromosomes for at least 20 million years. We sequenced the genomes of four species of Furcifer chameleons in the Illumina platform and compared the substitution rates of synonymous and non-synonymous mutations and their ratios among autosomal, Z-specific, and pseudoautosomal protein-coding genes. The inclusion of two chameleon outgroups lacking the differentiated ZZ/ZW sex chromosomes allowed us to control for gene-specific evolutionary rates that might confound the testing of the faster-X/Z effect. Significant differences in evolutionary rates were found between autosomal, Z-specific, and pseudoautosomal genes of Furcifer chameleons. However, the inclusion of the outgroups with different sex chromosomes suggests that these genes had different evolutionary rates prior to their incorporation into the differentiated ZZ/ZW sex chromosomes of the Furcifer genus. The results highlight the need to control for differences in the evolutionary rates of individual genes when testing for the faster X/Z effect.

Integrative Computational Framework, Dyscovr, Links Mutated Driver Genes to Expression Dysregulation Across 19 Cancer Types.

Though somatic mutations play a critical role in driving cancer initiation and progression, the systems-level functional impacts of these mutations-particularly, how they alter expression across the genome and give rise to cancer hallmarks-are not yet well-understood, even for well-studied cancer driver genes. To address this, we designed an integrative machine learning model, Dyscovr, that leverages mutation, gene expression, copy number alteration (CNA), methylation, and clinical data to uncover putative relationships between nonsynonymous mutations in key cancer driver genes and transcriptional changes across the genome. We applied Dyscovr pan-cancer and within 19 individual cancer types, finding both broadly relevant and cancer type-specific links between driver genes and putative targets, including a subset we further identify as exhibiting negative genetic relationships. Our work newly implicates-and validates in cell lines-KBTBD2 and mutant PIK3CA as putative synthetic lethals in breast cancer, suggesting a novel combinatorial treatment approach.

Estimation of PfRh5-based vaccine efficacy in asymptomatic Plasmodium falciparum patients from high-endemic areas of Tanzania using genetic and antigenicity variation screening.

Plasmodium falciparum is the most lethal malaria parasite. Recent phase 1b vaccine trials using P. falciparum reticulocyte binding protein homolog 5 (PfRh5) demonstrated safety and promising efficacy in preventing merozoite invasion. PfRh5 has emerged as a strong vaccine candidate due to its essential role in merozoite invasion and limited sequence variation. For effective malaria vaccine development, especially in high-transmission settings, strain-transcending activity must be considered. Ongoing monitoring of antigenic variation and natural immune responses is important to estimate vaccine efficacy across geographically diverse populations. Samples for this study were collected from four villages in each of the Kigoma and Geita regions, known malaria transmission hotspots in Tanzania. This community-based cross-sectional study was conducted from December 2022 to July 2023. Genetic variation and natural selection pressure on pfrh5 were analyzed in 164 asymptomatic P. falciparum isolates. The humoral immune response to PfRh5 was also assessed using a protein microarray with 242 sera samples from asymptomatic patients in the same population. Finally, a correlation analysis was conducted to compare pfrh5 genetic variation with the humoral immune response. The results revealed that pfrh5 was well conserved, but novel non-synonymous mutations were found at D65H, H170N, and I227M. Additionally, natural selection metrics indicated the potential for positive selection and a recent population expansion of PfRh5 in the study area, both of which could influence vaccine effectiveness. Antigenicity screening revealed variable sensitivity, ranging from 3.3% in Bunyambo to 82.8% in Rwantaba, with no significant relationship between antigenicity and parasitemia, haplotypes, or gender. However, age was significantly associated with humoral immune response (ρ = 0.170, p = 0.008). These findings underscore the need for future PfRh5-based vaccines to consider for increasing genetic variation and geographical differences in humoral immune responses.

Widespread geographic distribution of Aedes aegypti (Diptera: Culicidae) kdr variants in Panama.

We searched for evidence of knockdown resistance (kdr) mutations in the voltage-gated sodium channel gene of Aedes aegypti (Linnaeus) (Diptera: Culicidae) and Aedes albopictus (Skuse) (Diptera: Culicidae) mosquitoes from Panama. Conventional PCR was performed on 469 Ae. aegypti and 349 Ae. albopictus. We did not discover kdr mutations in Ae. albopictus, but 2 nonsynonymous kdr mutations, V1016I (found in 101 mosquitoes) and F1534C (found in 29 of the mosquitoes with the V1016I), were detected in Ae. aegypti. These kdr mutations were present in all specimens that were successfully sequenced for both IIS5-S6 and IIIS6 regions, which included samples collected from 8 of the 10 provinces of Panama. No other kdr mutations were found in Ae. aegypti, including V1016G, which has already been reported in Panama. Findings suggest that the V1016I-F1534C variant is prevalent in Panama, which might be related to the introduction and passive movement of mosquitoes as part of the used-tire trade. However, we cannot rule out the possibility that selection on de novo replacement of kdr mutations also partially explains the widespread distribution pattern of these mutations. These 2 ecological and evolutionary processes are not mutually exclusive, though, as they can occur in tandem. Research in Panama needs to calculate the genotypic and allelic frequencies of kdr alleles in local Ae. aegypti populations and to test whether some combinations confer phenotypic resistance or not. Finally, future studies will have to track the introduction and spreading of new kdr mutations in both Aedes species.

Serotypes, Antimicrobial Susceptibility, and Potential Mechanisms of Resistance Gene Transfer in Erysipelothrix rhusiopathiae Strains from Waterfowl in Poland.

Erysipelas is a significant problem in the waterfowl farming in Poland, and information on the characteristics of the Erysipelothrix rhusiopathiae strains causing this disease is limited. In this study, we determined the serotypes, antimicrobial susceptibility, and potential mechanisms of resistance gene transfer in E. rhusiopathiae isolates (n = 60) from domestic geese and ducks. We also developed a multiplex PCR for the detection of resistance genes. The antimicrobial susceptibility of the isolates was assessed using the broth microdilution method. Resistance genes, integrative conjugative element (ICE)-specific genes, phage-specific genes, and serotype determinants were detected by PCR. Multilocus sequence typing (MLST) was performed for selected resistant strains. The comparative analyses included 260 E. rhusiopathiae strains whose whole genome sequences (WGSs) are publicly available. E. rhusiopathiae isolates represented 7 serotypes, among which serotypes 5 (38.3%) and 1b (28.3%) were the most common. All strains were susceptible to β-lactams, and the vast majority of them were resistant to tetracycline (85%) and enrofloxacin (80%). The percentages of isolates resistant to other antimicrobials used ranged from 3.3% to 16.7%. Ten isolates (16.7%) were found to be multidrug resistant (MDR). The genotypic resistance profiles of the E. rhusiopathiae strains corresponded to their phenotypic resistance, and the amplification patterns obtained using the 10-plex PCR developed in this study were fully consistent with the results of single PCRs. The most prevalent resistance gene was tetM. In enrofloxacin-resistant strains, nonsynonymous mutations in the gyrA and parC genes were identified. The presence of ICE-specific genes was confirmed in resistant strains, and in MDR isolates of serotype 8 that represented sequence type (ST) 113, prophage DNA (Javan630-like) linked to the lsaE gene was additionally detected. The results indicate that β-lactam antibiotics should be the first choice for the treatment of waterfowl erysipelas in Poland. ICEs, including a transposon from the Tn916/Tn1545 family, and bacteriophages are most likely responsible for the transfer of resistance genes in E. rhusiopathiae.

Unveiling resistance expression profile to powdery mildew in wheat via Bulked Segregant RNA-Seq

BackgroundDeveloping wheat cultivars with durable resistance to powdery mildew, caused by Blumeria graminis f. sp. tritici (Bgt), is crucial for sustainable agriculture. The wheat genotype MYC exhibited high resistance to the Bgt isolate E09 at the seedling stage. Genetic analysis identified a recessive gene, temporarily named PmMYC, responsible for this resistance. Understanding the molecular mechanisms underlying this resistance is essential for advancing breeding programs.ResultsBulked Segregant RNA-Seq revealed numerous alternative splicing events generated following Bgt infection, suggesting powdery mildew may disrupt alternative splicing and affect immune responses. Gene Ontology (GO) analysis indicated significant enrichment of differentially expressed genes in “response to stimuli” and “immune system processes”, implying their roles in disease defense. BSR-Seq analysis identified two high-confidence candidate regions for PmMYC on chromosome 2B, spanning 40,451,950 − 102,426,703 bp and 421,707,046–449,840,516 bp. Within these intervals, 740 genes were identified, with nonsynonymous mutations in 46 genes in the parents and bulks. Real-time PCR showed distinct expression profiles in four genes in resistant MYC compared to susceptible Yannong 21. KEGG and COG analyses of differentially expressed genes in candidate intervals revealed enrichment in immune processes related to plant-pathogen interactions, confirming that PmMYC initiated a broad immune response to prevent Bgt invasion.ConclusionThe study identified key genetic intervals and genes involved in the resistance of wheat genotype MYC to Bgt. The identified genes, particularly those with altered expression profiles, could serve as valuable targets for breeding programs aimed at developing wheat cultivars with durable resistance to powdery mildew. These findings enhanced our understanding of plant-pathogen interactions and provided a foundation for future genetic and functional studies.

CTIM-18. HIGH LEVEL OF MUTATIONAL LOAD AND DIVERSITY OF TUMOR INFILTRATING T LYMPHOCYTES ARE FAVORABLE PROGNOSTIC MARKERS FOR CANCER SURVIVAL IN CHILDREN WITH RECURRENT MEDULLOBLASTOMA DURING ADOPTIVE CELLULAR THERAPY

Abstract BACKGROUND Adoptive cellular therapy (ACT) has shown remarkable efficacy in some patients with solid tumors, resulting in durable complete responses. A key mechanism behind this success is the targeting of neoantigens by T cells. There is culminating evidence that tumor-infiltrating lymphocytes (TILs) have a major effect on the clinical attributes of human cancer and can influence the tumor response to various therapy regimens. We have been sequencing RNA isolated from patient’s tumors to evaluate their mutational burden and identify tumor-specific neoantigens. Additionally, we have identified the entire repertoire of tumor-infiltrating lymphocytes in patients with recurrent medulloblastoma. METHODS Total tumor RNA was extracted from resected brain tumor specimens and after we applied in-house bulk RNA-Seq and TCR-Seq techniques using rapid amplification of cDNA ends (RACE) technology with the addition of a common adapter at the 5’ end. RESULTS & CONCLUSIONS Analysis of the 22 tumor tissues revealed that the frequency of non-synonymous mutations varied dramatically more than 10-fold among patients with recurrent medulloblastoma undergoing adoptive cellular therapy (Re-MATCH protocol, FDA IND BB-14058). Patients with shorter overall survival exhibited non-synonymous mutation frequencies between 0.113 and 0.335 mutations per megabase (Mb). Children with prolonged overall survival had higher mutation frequencies, ranging from 0.6 to 1.1 mutations per Mb. These findings highlight the potential of using mutational burden as a biomarker for patient selection and treatment customization, ultimately aiming to improve outcomes in ACT for recurrent medulloblastoma. Up to 2000 unique TCR clones were found among TIL populations in patients who responded well to ACT. Patients with poor outcomes had significantly lower TCR diversity, with only 10-40 unique TCR clones among TILs. The correlation between TCR diversity among TILs and clinical outcomes in pediatric brain tumors underscores the importance of a diverse T cell repertoire for the success of ACT.

IMMU-40. INVESTIGATING THE ROLE OF MUTATIONAL BURDEN IN TUMOR IMMUNOGENICITY USING A PRE-CLINICALMLH1-DEFICIENT GLIOBLASTOMA MODEL

Abstract BACKGROUND Although there is an established correlation between tumor mutational burden (TMB) and response to immune checkpoint inhibition (ICI) in many cancers, such an association has not been definitively shown in high-grade gliomas. Most studies of ICI therapy in gliomas, including glioblastoma (GBM), have failed to demonstrate efficacy, with the exception of hypermutated cases resulting from rare inherited defects in mismatch repair (MMR) genes. We sought to investigate the association of TMB to immune response using a syngeneic murine model with a spectrum of mutational burdens. METHODS We used CRISPR-Cas9 to knockout the MMR gene Mlh1 in the murine glioma model SB28, an ICI-resistant cell line with a very low mutational burden (108 non-synonymous mutations). RESULTS Single-cell sorted Mlh1 knockout SB28 clones revealed a two- to six-fold increase in TMB. Importantly, loss of Mlh1 also brought about global changes in immune- and inflammatory-related gene expression in vitro, without correlation to increasing TMB. While all clones had the same rate of in vitro proliferation, flank injection of a high TMB vs a low TMB SB28 Mlh1 knockout clone resulted in similar survival benefits compared to Mlh1 wildtype SB28. CONCLUSIONS These preliminary results suggest that, although disruption of the MMR system may result in heightened tumor immunogenicity, elevated TMB may not be the sole significant determinant. We are currently investigating tumor proliferation after flank injection in immunocompromised mice to confirm that slowed growth is immune-related, as well as clone-specific response to ICI treatment. We are also exploring the role of specific cytokines/chemokines in conferring this survival advantage and potential immune response. By generating a GBM model of varied mutational burdens and immune profiles and comparing to the baseline SB28 cell line, we can interrogate the relative significance of TMB in tumor immunogenicity and ICI response.

Genomic Surveillance and Molecular Characterization of SARS-CoV-2 Variants During the Peak of the Pandemic in Türkiye.

SARS-CoV-2 is a highly transmissible coronavirus and has caused a pandemic of acute respiratory disease. Genomic characterization of SARS-CoV-2 is important for monitoring and assessing its evolution. A total of 1.346 nasopharyngeal swab samples were collected but only 879 SARS-CoV-2 high-quality genomes were isolated, subjected to Next Generation Sequencing and analyzed both statistically and regarding mutations comprehensively. The distribution of clades and lineages in different cities of Türkiye and the association of SARS-CoV-2 variants with age groups and clinical characteristics of COVID-19 were also examined. Furthermore, the frequency of the clades and lineages was observed in 10months. Finally, non-synonymous mutations not defined in specific SARS-CoV-2 variants (during that period) were identified by performing mutation analysis. B.1.1.7 (Alpha) and B.1.617.2 (Delta) SARS-CoV-2 variants which have also been identified in our study from March to December 2021. We observed a significant association of SARS-CoV-2 variants with age groups and cities. Also, E:T9I, S:A27S, S:A67V, S:D796Y, S:K417N, S:N440K, S:R158X, S:S477N (below 1%-frequency) were determined as specific mutations belonging and shared with the Omicron variant that appeared later. Our study has highlighted the importance of constant monitoring of the genetic diversity of SARS-CoV-2 to provide better prevention strategies and it contributes to the understanding of SARS-CoV-2 from the past to the present.

Association of multilocus sequence typing, MSH2 gene mutations, and antifungal resistance in Candida glabrata: implications for clinical outcomes in Chinese hospitals

BackgroundCandida glabrata is the second most common cause of invasive candidiasis worldwide. In this study, we determined the clinical characteristics and drug sensitivity of C. glabrata isolates and investigated the associations between MSH2 gene mutations, sequence types (ST), and drug resistance.MethodsA total of 154 C. glabrata isolates were collected from patients being treated in three hospitals in China. The antifungal sensitivity of the strains was assessed using the broth microdilution method. Multilocus sequence typing (MLST) was also performed, followed by MSH2 sequencing. The clinical features and outcomes of C. glabrata infection were analysed for a total of 49 strains, which were collected from patients with invasive Candida infection at Longhua Hospital.ResultsAll 154 isolates were found to be susceptible to amphotericin, 5-fluorocytosine, anidulafungin, caspofungin, and micafungin, whereas 11.7% were fluconazole-resistant, 18.8% were itraconazole non-wild type, and 35.7% were voriconazole non-wild type. ST7 (62.34%) was the most common ST genotype, followed by ST10 (16.88%) and ST15 (7.79%). The total azole resistance rates for all isolates, ST7, ST10, and other STs were 36.4, 42.7, 34.6, and 18.8%, respectively. The ST7 and ST10 isolates were characterised by a higher drug resistance rate than the other minor ST isolates. Moreover, 59.09% of isolates had one or more MSH2 non-synonymous mutations, with V239L being the most commonly detected mutation. The frequency of MSH2 mutations was significantly higher in azole-resistant isolates than in other isolates, whereas P6L or L87P mutations were associated with the highest azole resistance rates of up to 87.5% and 80%, respectively. Our results indicated that ST7 and ST15 are independent predictors of mortality caused by C. glabrata infection and revealed a higher 30-day mortality in patients infected with these strains than in those infected with other ST isolates.ConclusionsOur findings revealed the relationships between MLST, MSH2 gene mutations, and drug resistance in the common pathogenic fungus C. glabrata, and thereby enabled us to identify strains that are associated with higher rates of mortality. These findings will contribute to enhancing our understanding of the pathogenesis of C. glabrata infection.

Enhanced detection and molecular modeling of adaptive mutations in SARS-CoV-2 coding and non-coding regions using the c/µ test.

Accurately identifying mutations under beneficial selection in viral genomes is crucial for understanding their molecular evolution and pathogenicity. Traditional methods like the Ka/Ks test, which assesses non-synonymous (Ka) versus synonymous (Ks) substitution rates, assume that synonymous substitutions at synonymous sites are neutral and thus is equal to the mutation rate (µ). Yet, evidence suggests that synonymous sites in translated regions (TRs) and untranslated regions (UTRs) can be under strong beneficial selection (Ks > µ) and strongly conserved (Ks ≈ 0), leading to false predictions of adaptive mutations from codon-by-codon Ka/Ks analysis. Our previous work used a relative substitution rate test (c/µ, c: substitution rate in UTR/TR, and µ: mutation rate) to identify adaptive mutations in SARS-CoV-2 genome without the neutrality assumption of the synonymous sites. This study refines the c/µ test by optimizing µ value, leading to a smaller set of nucleotide and amino acid sites under beneficial selection in both UTR (11 sites with c/µ > 3) and TR (69 nonsynonymous sites: c/µ > 3 and Ka/Ks > 2.5; 107 synonymous sites: Ks/µ > 3). Encouragingly, the top two mutations in UTR and 70% of the top nonsynonymous mutations in TR had reported or predicted effects in the literature. Molecular modeling of top adaptive mutations for some critical proteins (S, NSP11, and NSP5) was carried out to elucidate the possible molecular mechanism of their adaptivity.