Number Of Single Nucleotide Variants Research Articles

Molecular cloning of PRD-like homeobox genes expressed in bovine oocytes and early IVF embryos.

Embryonic genome activation (EGA) is a critical step in early embryonic development, as it marks the transition from relying on maternal factors to the initiation of transcription from embryo's own genome. The factors associated with EGA are not well understood and need further investigation. PRD-like (PRDL) homeodomain transcription factors (TFs) are considered to play crucial roles in this early event during development but these TFs have evolved differently, even within mammalian lineages. Different numbers of PRDL TFs have been predicted in bovine (Bos taurus); however, their divergent evolution requires species-specific confirmation and functional investigations. In this study, we conducted molecular cloning of mRNAs for the PRDL TFs ARGFX, DUXA, LEUTX, NOBOX, TPRX1, TPRX2, and TPRX3 in bovine oocytes or in vitro fertilized (IVF) preimplantation embryos. Our results confirmed the expression of PRDL TF genes in early bovine development at the cDNA level and uncovered their structures. For each investigated PRDL TF gene, we isolated at least one homeodomain-encoding cDNA fragment, indicative of DNA binding and thus potential role in transcriptional regulation in developing bovine embryos. Additionally, our cDNA cloning approach allowed us to reveal breed-related differences in bovine, as evidenced by the identification of a high number of single nucleotide variants (SNVs) across the PRDL class homeobox genes. Subsequently, we observed the prediction of the 9aa transactivation domain (9aaTAD) motif in the putative protein sequence of TPRX3 leading us to conduct functional analysis of this gene. We demonstrated that the TPRX3 overexpression in bovine fibroblast induces not only protein-coding genes but also short noncoding RNAs involved in splicing and RNA editing. We supported this finding by identifying a shared set of genes between our and published bovine early embryo development datasets. Providing full-length cDNA evidence for previously predicted homeobox genes that belong to PRDL class improves the annotation of the bovine genome. Updating the annotation with seven developmentally-important genes will enhance the accuracy of RNAseq analysis with datasets derived from bovine preimplantation embryos. In addition, the absence of TPRX3 in humans highlights the species-specific and TF-specific regulation of biological processes during early embryo development.

Searching for genetic determinants for left ventricular non-compaction.

Left ventricular non-compaction (LVNC) is still a pathology around which there are numerous controversies regarding the criteria for its diagnosis, presentation, prognosis, and even classification into the appropriate group of diseases. So far, about 190 genes in which mutations may be associated with LVNC have been described, and in each of them, several to several dozen different loci have been discovered. We decided to analyze the frequency of single nucleotide variants (SNVs) in correlation to Petersen's criteria. We retrospectively analyzed the results of cardiac magnetic resonance (CMR) studies. Twenty-three patients who met Petersen's criteria agreed to participate in the research and take blood samples for genetic testing. Next, we prospectively included 24 volunteers who did not meet Petersen's criteria. Petersen's criteria were complied with ratio of non-compacted to compacted myocardium (NC/C) ≥2.3. A total of 47 DNA samples were analyzed based on the selected regions of the following genes: β-myosin heavy chain (MYH7), α-cardiac actin (ACTC1), cardiac troponin T (TNNT2), myosin binding protein-C (MYBPC3), LIM-domain binding protein 3 (LBD3), and taffazin (TAZ). In total, 248 substitutions in exons and introns were obtained for all analyzed samples. No statistically significant differences were detected between the mentioned groups. No significant difference in either downward or upward trends in the number of substitutions in relation to the increasing trabeculation is observed. We indicated differences in the occurrence of the studied SNVs between groups, especially for rs8037241 (3'UTR region of ACTC1) and rs2675686 (LDB3), but they also did not show statistical significance. Although we did not find a significant correlation between the co-occurrence of individual mutations with LVNC, it is worth noting that the presence of one of the four mutations in the range rs8037241 (ACTC1 3'UTR), rs3729998 (TNNT2e. 12), and rs727503240 (MYH7e. 39) increases the risk of LVNC more than 4 times. An inverse association between the number of SNVs and the meeting the Petersen's criteria was demonstrated for studied LDB3 region and rs397516254 in exon 39 of the MYH7 gene. To our knowledge, no studies have been published comparing the prevalence of selected SNVs in a group of healthy subjects and in a group meeting the Petersen criteria for LVNC. Among both completely healthy individuals who did not meet the Petersen criteria for LVNC as well as those with symptoms who met these criteria we found a similar incidence of SNVs in the ACTC1, TNNT2, LDB3 and MYH7 genes segments analyzed. Further studies are required to confirm or exclude "potentially protective" SNV in the 39th exon of MYH7 (rs397516254) and the role of co-occurrence of individual SNVs in rs8037241 (ACTC1 3'UTR), rs3729998 (TNNT2), and rs727503240 (MYH7) for the increase of the risk of LVNC.

The hare syphilis agent is related to, but distinct from, the treponeme causing rabbit syphilis.

The treponemes infecting lagomorphs include Treponema paraluisleporidarum ecovar Cuniculus (TPeC) and ecovar Lepus (TPeL), infecting rabbits and hares, respectively. In this study, we described the first complete genome sequence of TPeL, isolate V3603-13, from an infected mountain hare (Lepus timidus) in Sweden. In addition, we determined 99.0% of the genome sequence of isolate V246-08 (also from an infected mountain hare, Sweden) and 31.7% of the genome sequence of isolate Z27 A77/78 (from a European hare, Lepus europeaus, The Netherlands). The TPeL V3603-13 genome had considerable gene synteny with the TPeC Cuniculi A genome and with the human pathogen T. pallidum, which causes syphilis (ssp. pallidum, TPA), yaws (ssp. pertenue, TPE) and endemic syphilis (ssp. endemicum, TEN). Compared to the TPeC Cuniculi A genome, TPeL V3603-13 contained four insertions and 11 deletions longer than three nucleotides (ranging between 6 and2,932 nts). In addition, there were 25 additional indels, from one to three nucleotides long, altogether spanning 36 nts. The number of single nucleotide variants (SNVs) between TPeC Cuniculi A and TPeL V3603-13 were represented by 309 nucleotide differences. Major proteome coding differences between TPeL and TPeC were found in the tpr gene family, and (predicted) genes coding for outer membrane proteins, suggesting that these components are essential for host adaptation in lagomorph syphilis. The phylogeny revealed that the TPeL sample from the European brown hare was more distantly related to TPeC Cuniculi A than V3603-13 and V246-08.

Genome-wide association study and candidate gene identification for agronomic traits in 182 upward-growing fruits of C. frutescens and C. annuum

Pepper agronomic traits serve as pivotal indicators for characterizing germplasm attributes and correlations. It is important to study differential genotypic variation through phenotypic differences of target traits. Whole genome resequencing was used to sequence the whole genome among different individuals of species with known reference genomes and annotations, and based on this, differential analyses of individuals or populations were carried out to identify SNPs for agronomic traits related to pepper. This study conducted a genome-wide association study encompassing 26 key agronomic traits in 182 upward-growing fruits of C. frutescens and C. annuum. The population structure (phylogenetics, population structure, population principal component analysis, genetic relationship) and linkage disequilibrium analysis were realized to ensure the accuracy and reliability of GWAS results, and the optimal statistical model was determined. A total of 929 SNPs significantly associated with 26 agronomic traits, were identified, alongside the detection of 519 candidate genes within 100 kb region adjacent to these SNPs. Additionally, through gene annotation and expression pattern scrutiny, genes such as GAUT1, COP10, and DDB1 correlated with fruit traits in Capsicum frutescens and Capsicum annuum were validated via qRT-PCR. In the CH20 (Capsicum annuum) and YB-4 (Capsicum frutescens) cultivars, GAUT1 and COP10 were cloned with cDNA lengths of 1065 bp and 561 bp, respectively, exhibiting only a small number of single nucleotide variations and nucleotide deletions. This validation provides a robust reference for molecular marker-assisted breeding of pepper agronomic traits, offering both genetic resources and theoretical foundations for future endeavors in molecular marker-assisted breeding for pepper.

Whole-Genome Sequencing and Analysis of Tumour-Forming Radish (Raphanus sativus L.) Line.

Spontaneous tumour formation in higher plants can occur in the absence of pathogen invasion, depending on the plant genotype. Spontaneous tumour formation on the taproots is consistently observed in certain inbred lines of radish (Raphanus sativus var. radicula Pers.). In this paper, using Oxford Nanopore and Illumina technologies, we have sequenced the genomes of two closely related radish inbred lines that differ in their ability to spontaneously form tumours. We identified a large number of single nucleotide variants (amino acid substitutions, insertions or deletions, SNVs) that are likely to be associated with the spontaneous tumour formation. Among the genes involved in the trait, we have identified those that regulate the cell cycle, meristem activity, gene expression, and metabolism and signalling of phytohormones. After identifying the SNVs, we performed Sanger sequencing of amplicons corresponding to SNV-containing regions to validate our results. We then checked for the presence of SNVs in other tumour lines of the radish genetic collection and found the ERF118 gene, which had the SNVs in the majority of tumour lines. Furthermore, we performed the identification of the CLAVATA3/ESR (CLE) and WUSCHEL (WOX) genes and, as a result, identified two unique radish CLE genes which probably encode proteins with multiple CLE domains. The results obtained provide a basis for investigating the mechanisms of plant tumour formation and also for future genetic and genomic studies of radish.

HiChIP-Based Epigenomic Footprinting Identifies a Promoter Variant of UXS1 That Confers Genetic Susceptibility to Gastroesophageal Cancer.

Genome-wide association studies (GWAS) have identified more than a hundred single nucleotide variants (SNV) associated with the risk of gastroesophageal cancer (GEC). The majority of the identified SNVs map to noncoding regions of the genome. Uncovering the causal SNVs and genes they modulate could help improve GEC prevention and treatment. Herein, we used HiChIP against histone 3 lysine 27 acetylation (H3K27ac) to simultaneously annotate active promoters and enhancers, identify the interactions between them, and detect nucleosome-free regions (NFR) harboring potential causal SNVs in a single assay. The application of H3K27ac HiChIP in GEC relevant models identified 61 potential functional SNVs that reside in NFRs and interact with 49 genes at 17 loci. The approach led to a 67% reduction in the number of SNVs in linkage disequilibrium at these 17 loci, and at 7 loci, a single putative causal SNV was identified. One SNV, rs147518036, located within the promoter of the UDP-glucuronate decarboxylase 1 (UXS1) gene, seemed to underlie the GEC risk association captured by the rs75460256 index SNV. The rs147518036 SNV creates a GABPA DNA recognition motif, resulting in increased promoter activity, and CRISPR-mediated inhibition of the UXS1 promoter reduced the viability of the GEC cells. These findings provide a framework that simplifies the identification of potentially functional regulatory SNVs and target genes underlying risk-associated loci. In addition, the study implicates increased expression of the enzyme UXS1 and activation of its metabolic pathway as a predisposition to gastric cancer, which highlights potential therapeutic avenues to treat this disease. Significance: Epigenomic footprinting using a histone posttranslational modification targeted 3D genomics methodology elucidates functional noncoding sequence variants and their target genes at cancer risk loci.

Abstract IA016: Molecular diversity of uterine serous carcinoma

Abstract Uterine serous carcinoma (USC) is a biologically aggressive subtype of endometrial cancer with a high recurrence rate and poor prognosis disproportionately affecting black women. Whole-exome-sequencing analysis revealed that up to 10% of USC harbor features of ultra/hypermutation due to a deficiency of polymerase e (POLE) and/or mismatched-repair genes (MMRd). These tumors are notable for having a high number of single nucleotide variants (SNV), no LOH segments or copy-number variants (CNV) and a peculiar genetic signature characterized by an increased rate of C > A transversions. In contrast, the overwhelming majority of USC (~ 90%) are POLE/MMR proficient, have a low number of SNV and demonstrate alteration in genes including TP53, PIK3CA mutation/amplification, ERBB2 amplification, CCNE1 amplification, FBXW7 mutation/deletion, PPP2R1A mutation, and somatic mutations involving homologous recombination genes. Frequent mutations in CHD4/Mi2b, a member of the NuRD-chromatin-remodeling-complex, and TAF1, an element of the core TFIID transcriptional machinery are also detected in a significant number of USC. Somatic CNVs, a sign of genomic instability, are identified in the majority of USC with multiple copy number gains and copy number losses, with the most frequent events taking place in pathways similar to those found from analysis of SNVs. In addition to loss of TP53, frequent deletion of a small segment of chromosome 19 containing MBD3, a member of the NuRD-chromatin-modification-complex, and frequent amplification of chromosome segments containing PIK3CA, ERBB2 (encoding for the HER2/neu receptor) and CCNE1 (a target of FBXW7-mediated ubiquitination) are commonly detected. Overall, these findings suggest that both somatic point mutations (SNV) and somatic CNV play a major role in the pathogenesis of USC. Importantly, the frequent mutation of DNA damage, chromatin remodeling, cell cycle and cell proliferation pathways in USC including the overexpression/amplification of HER2/neu, CCNE1 and the tumor-associated calcium signal transducer 2 (TACSTD2/TROP2) genes, suggest novel targets for therapy against this lethal variant of endometrial cancer. Citation Format: Alessandro D. Santin. Molecular diversity of uterine serous carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Endometrial Cancer: Transforming Care through Science; 2023 Nov 16-18; Boston, Massachusetts. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(5_Suppl):Abstract nr IA016.

Comparison of the DNBSEQ platform and Illumina HiSeq 2000 for bacterial genome assembly

The Illumina HiSeq platform has been a commonly used option for bacterial genome sequencing. Now the BGI DNA nanoball (DNB) nanoarrays platform may provide an alternative platform for sequencing of bacterial genomes. To explore the impact of sequencing platforms on bacterial genome assembly, quality assessment, sequence alignment, functional annotation, mutation detection, and metagenome mapping, we compared genome assemblies based on sequencing of cultured bacterial species using the HiSeq 2000 and BGISEQ-500 platforms. In addition, simulated reads were used to evaluate the impact of insert size on genome assembly. Genome assemblies based on BGISEQ-500 sequencing exhibited higher completeness and fewer N bases in high GC genomes, whereas HiSeq 2000 assemblies exhibited higher N50. The majority of assembly assessment parameters, sequences of 16S rRNA genes and genomes, numbers of single nucleotide variants (SNV), and mapping to metagenome data did not differ significantly between platforms. More insertions were detected in HiSeq 2000 genome assemblies, whereas more deletions were detected in BGISEQ-500 genome assemblies. Insert size had no significant impact on genome assembly. Taken together, our results suggest that DNBSEQ platforms would be a valid substitute for HiSeq 2000 for bacterial genome sequencing.

Transcriptomic and genomic profiling of multiple primary colorectal cancers reveals intratumor heterogeneity and a distinct immune microenvironment

This study reported on the intratumor genomic and immunological heterogeneity of different tumor lesions from a single patient with multiple primary colorectal cancer (MPCC). The goal of this study was to explore the molecular and microenvironment characteristics of tumor lesions from different primary sites in a patient with MPCC.A total of three tumor lesions located in the hepatic flexure of the transverse colon, sigmoid colon, and rectum were collected from a 72-year-old male patient with MPCC. All three tumor samples were examined by using whole-exome sequencing (WES) and single-cell RNA sequencing (scRNA-seq). The transcriptome data of The Cancer Genome Atlas (TCGA) colon cancer (COAD) dataset were explored to characterize the biological impacts of certain immune cells. Only three nonsynonymous mutations were shared by all of the tumor lesions, whereas a number of single nucleotide variant (SNV) and copy number variation (CNV) mutations were shared by tumor samples from the sigmoid colon and rectum. Transcriptomic analysis showed that tumor lesions derived from the transverse colon had decreased levels of RTK, ERK, and AKT pathway activity, thus suggesting lower oncogenic properties in the transverse lesion compared to the other two samples. Further immune landscape evaluation by using single-cell transcriptomic analysis displayed significant intratumor heterogeneity in MPCC. Specifically, more abundant mucosal-associated invariant T (MAIT) cell infiltration was found in transverse colon tumor lesions. Afterwards, we found that higher MAIT cell infiltration may correlate with a better prognosis of patients with colon cancer (immunohistochemical status was MSI-L/pMMR) by using a publicly available TCGA dataset.

Impact of Genomic Alterations on the Clinical Outcome of Patients With Hepatitis B-related Hepatocellular Carcinoma Receiving Curative Surgery: A Retrospective Cohort Study.

The aim of the current study was to obtain comprehensive genomic information on viral hepatitis B (HBV)-related hepatocellular carcinoma (HCC) and identify potential biomarkers of early recurrence in patients receiving curative surgery. A total of 104 patients with HBV-related HCC receiving curative surgery at Kaohsiung Chang Gung Memorial Hospital between January 2017 and December 2020 were identified, including 52 patients each with and without recurrence. Next-generation sequencing was performed to investigate genomic alterations caused by surgical resection of specimens. The Kaplan-Meier method was used to estimate disease-free survival and overall survival. The landscape of gene mutations in HCC patients of our cohort showed a median number of single nucleotide variants of 250, a median number of insertions and deletions of 22, and a median number of protein-coding mutations of 185. The 10 most frequently mutated genes were TP53 (43%), TTN (39%), MUC16 (28%), PCLO (25%), OBSCN (22%), ADGRV1 (19%), ALB (18%), SYNE1 (18%), DNAH17 (17%), and RYR1 (17%). The tumour mutation burden was 4.8 mutations per megabase, and high microsatellite instability was reported in only three patients. In addition, the mutational signatures showed that aristolochic acid exposure was highly implicated in our HCC cohort. Five mutant genes, TBC1D4, ITGA4, RPS6KA3, VWA8, and FMN2, were more frequent in the recurrence group than that in the non-recurrence group. Our results present an in-depth genomic analysis of HBV-related HCC. The study findings provide an improved understanding of the related molecular mechanisms and identify potential biomarkers associated with early tumour recurrence after curative resection.

Abstract PO-075: Immunosuppression allows for proliferation of alternative splicing events in a murine model of tobacco-associated oral cavity squamous cell carcinoma

Abstract Background: Non-canonical alternative splicing events have been demonstrated to occur in head and neck squamous cell carcinoma, and result in functionally active aberrant isoforms that contribute to carcinogenesis. Our preliminary data also demonstrates that high splicing burden is associated with higher recurrence rates in oral cancer as well as relative immunosuppression. These splicing events are hypothesized to generate immunogenic neoantigens given non-self sequences that are generated from these novel isoforms. However, the role of the immune system in regulating the proliferation of aberrant splicing is not well understood. Therefore, we evaluated splicing alteration in the setting of immunosuppression in a murine model of oral cancer. Study Design: An established murine cell line with genetic signature resembling human tobacco-associated oral cavity SCC (4MOSC1) was injected into the oral tongue of wild type (C57/Blk6) and immunocompromised (SCID) mice. At takedown, specimens were collected for bulk RNA sequencing (RNASeq). Fastq files were aligned to mm39 using STAR v.2.7.1a. Splicing analysis was performed using OutSplice. Somatic variants in tumor samples were determined from RNASeq data using the GATK v4.4.0 tool Mutect2. Mutational burden was estimated using the number of single nucleotide variants (SNVs) called in each sample (CountVariants, GATK). Trinity v2.12.0 and NetMHCPan v4.1 were used to define PHBR scores to predict MHC binding affinity of neopeptides. Results: Seven of ten mice had measurable tumor by day 5, for an engraftment rate of 70% (3/5 C57/Blk6; 4/5 SCID). At day 15, tumors in wild type mice had a mean volume of 14.7mm3 compared to 28.9mm3 in SCID mice (one-sided p=0.132). Sequencing analysis demonstrated that splicing burden was higher in tumors developed in SCID mice compared to Blk6 mice (mean 475.25 vs 381.67, p=0.022). The mean number of SNVs were 144.2 mt/mb in SCID mice compared to 81.2 mt/mb in Blk6 mice (p=0.044). Amongst predicted splice derived neopeptides, those in SCID tumors had lower PHBR scores versus wild type tumors (median: 3.13 vs 4.23, p<0.001), correlating with higher predicted MHC binding affinity. However, amongst very strong binders (PHBR score ≤0.5), there was no difference in PHBR score between groups (0.30 vs 0.22, p=0.97). Conclusion: As expected, immunosuppression allowed for increased growth of tumors in the murine oral cancer model. We show that immunosuppression allows for the proliferation of both alternative splicing events and mutations. These preliminary data demonstrate that the immune system may play a role in regulating aberrant splicing events in oral cancer. Aberrant splice derived peptides which stimulate the immune system may be more likely to develop in an immunosuppressed setting. Citation Format: Mitchell Victor, Sophie Jang, Joseph Bendik, Silvio Gutkind, Theresa Guo. Immunosuppression allows for proliferation of alternative splicing events in a murine model of tobacco-associated oral cavity squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PO-075.

Transcriptomic Analysis of Medicago truncatula under Long-Day Conditions

The objective of this research was to understand the expression characteristics and biological functions of Medicago truncatula genes under long-day conditions. The leaves of “R108” tribulus Medicago truncatula at the branch stage (A), bud stage (B), initial flowering stage (C), and full flowering stage (D) were sequenced using RNA-Seq technology. The genome of Medicago truncatula, a related species of Medicago truncatula, was used as the reference genome for sequence comparison. The transcriptomes of three adjacent periods (A vs. B, B vs. C, and C vs. D) were analyzed for differential gene expression and these genes were screened. A total of 6875 differentially expressed genes were detected. GO functional analysis showed that differentially expressed genes were mainly involved in biological processes, cell components, and molecular functions, among which the most differentially expressed genes were involved in the synthesis of cell components. KEGG enrichment analysis showed that the differentially expressed genes were mainly involved in circadian rhythm, photosynthetic antenna protein, ribosome metabolism, and other pathways. The number of single nucleotide variants detected by cSNP analysis was 312,875, and the frequency of A/G and C/T were the highest. The function of eggNOG was divided into 23 categories, with a total of 26,745 genes having similarities, while 9008 genes were classified as having an unknown function, 2669 genes were classified as part of signal transduction mechanisms, and 2194 genes were classified as being involved in transcription. In different developmental stages (A vs. B, B vs. C, and C vs. D), 3463 up-regulated and 3412 down-regulated differentially expressed genes were found. The difference between up-regulated and down-regulated genes was more noteworthy at the bud stage and the initial flowering stage. In addition, a total of 79 flowering genes were found, of which 51 differential genes were identified as participating in the photoperiodic regulation pathway, consisting of 23 differential genes that were up-regulated, and 28 differential genes that were down-regulated. The ratios of gene-LOC11410562(GI), gene-LOC11435974(CO), gene-LOC11422615(TOC1), and gene-LOC11432385(LHY) were higher than those of gene-LOC25500742(PHYA) and gene-LOC11 431402(ELF3); gene-LOC11434778(Col13), gene-LOC25498015(Col6), and gene-LOC11415514(Col9) were pre-expressed. The above differentially expressed genes were significantly expressed in different developmental stages of Medicago truncatula, which lays a foundation for further study of the molecular mechanism of Medicago truncatula.

ZFP37, C3H, NAC94, and bHLH148 transcription factors regulate cultivar-specific drought response by modulating r40C1 gene expression in rice

Lectins are gradually gaining importance in regulating abiotic stress response in plants. Among them, the R40 family plays pivotal role in regulating drought response. The expression of the Osr40C1 positively correlates with the degree of drought tolerance in indica rice cultivars. Although the mechanism of Osr40C1-mediated drought tolerance is well characterized, the gene's regulation under drought remains obscure. Here, we demonstrate that the Osr40C1 promoter is activated under drought to induce its transcription. A number of single nucleotide variations in the Osr40C1 promoter of the drought-tolerant IR36 and sensitive MTU1010 cultivar are detected. However, promoter swapping analysis reveals that these variations in the promoter sequence do not explain the cultivar-specific expression pattern of this gene. Next, we identified the transcription factors that interact with the Osr40C1 promoter. Among these transcription factors, OsZFP37, OsC3H, OsNAC94, and OsbHLH148 display significantly higher transcript abundance in IR36 compared with MTU1010. Furthermore, overexpression of each of these transcription factors confers drought tolerance, whereas their silencing leads to drought sensitivity. Interestingly, the Osr40C1 expression is significantly up-regulated in the transcription factor overexpression lines, while its expression is undetectable in the silencing lines. Together our study highlights how different transcription factors regulate the cultivar-specific expression pattern of Osr40C1 in rice.

Characterizing the role of Phlda3 in the development of acute toxicity and malignant transformation of hematopoietic cells induced by total-body irradiation in mice

The tumor suppressor p53 is a transcriptional factor that plays a crucial role in controlling acute toxicity and long-term malignant transformation of hematopoietic cells induced by genotoxic stress such as ionizing radiation. Among all transcriptional targets of p53, one gene that is robustly induced by radiation is the pleckstrin homology domain-only protein Phlda3. However, the role that Phlda3 plays in regulating the response of hematopoietic cells to radiation is unknown. Here, using isogenic cell lines and genetically engineered mouse models, we showed that radiation induces Phlda3 in human leukemia cells and mouse normal hematopoietic cells in a p53-dependent manner. However, deletion of the Phlda3 gene did not ameliorate radiation-induced acute hematologic toxicity. In addition, distinct from mice that lose p53, loss of Phlda3 did not alter the latency and incidence of radiation-induced thymic lymphoma in mice. Remarkably, whole-exome sequencing data showed that lymphomas in irradiated Phlda3+/+ mice harbor a significantly higher number of single nucleotide variants (SNVs) and indels compared to lymphomas in irradiated Phlda3+/− and Phlda3−/− littermates. Together, our results indicate that although deletion of Phlda3 does not accelerate the development of radiation-induced thymic lymphoma, fewer SNVs and indels are necessary to initiate lymphomagenesis after radiation exposure when Phlda3 is silenced.

Critically short telomeres derepress retrotransposons to promote genome instability in embryonic stem cells

Telomeres, at the ends of chromosomes, protect chromosomes from fusion and preserve genomic stability. However, the molecular mechanisms underlying telomere attrition-induced genome instability remain to be understood. We systematically analyzed the expression of retrotransposons and performed genomic sequencing of different cell and tissue types with telomeres of varying lengths due to telomerase deficiency. We found that critically short telomeres altered retrotransposon activity to promote genomic instability in mouse embryonic stem cells, as evidenced by elevated numbers of single nucleotide variants, indels and copy number variations (CNVs). Transpositions of retrotransposons such as LINE1 resulting from the short telomeres can also be found in these genomes with elevated number of mutations and CNVs. Retrotransposon activation is linked to increased chromatin accessibility, and reduced heterochromatin abundance correlates with short telomeres. Re-elongation of telomeres upon recovery of telomerase partly represses retrotransposons and heterochromatin accumulation. Together, our findings suggest a potential mechanism by which telomeres maintain genomic stability by suppressing chromatin accessibility and retrotransposon activity.

Multimarker omnibus tests by leveraging individual marker summary statistics from large biobanks.

As biobanks become increasingly popular, access to genotypic and phenotypic data continues to increase in the form of precomputed summary statistics (PCSS). Widespread accessibility of PCSS alleviates many issues related to biobank data, including that of data privacy and confidentiality, as well as high computational costs. However, questions remain about how to maximally leverage PCSS for downstream statistical analyses. Here we present a novel method for testing the association of an arbitrary number of single nucleotide variants (SNVs) on a linear combination of phenotypes after adjusting for covariates for common multimarker tests (e.g., SKAT, SKAT-O) without access to individual patient-level data (IPD). We validate exact formulas for each method, and demonstrate their accuracy through simulation studies and an application to fatty acid phenotypic data from the Framingham Heart Study.

Echoed induction of nucleotide variants and chromosomal structural variants in cancer cells

Generally, the number of single-nucleotide variants (SNVs) in somatic cells increases with age, which is expected for replication errors. The number of SNVs in cancer cells, however, is often much higher than that in somatic cells, raising the question of whether cancer cells possess SNV induction pathways. The present study shows that the number of SNVs in cancer cells correlates with the number of chromosomal structural variants (SVs). While Kataegis, localized hypermutations typically arising near SV sites, revealed multiple SNVs within 1 kb, SV-associated SNVs were generally observed within 0.1–1 Mb of SV sites, irrespective of Kataegis status. SNVs enriched within 1 Mb of SV regions were associated with deficiency of DNA damage repair, including HR deficiency-associated single base substitution 3 (SBS3) and exogenous damage-associated SBS7 and SBS36 signatures. We also observed a similar correlation between SVs and SNVs in cells that had undergone clonal evolution in association with genomic instability, implying an association between genomic instability and SV-associated induction of SNVs.

The ctDNA-based postoperative molecular residual disease status in different subtypes of early-stage breast cancer.

Breast cancer is a highly heterogeneous disease. Early-stage, non-metastatic breast cancer is considered curable after definitive treatment. Early detection of tumor recurrence and metastasis through sensitive biomarkers is helpful for guiding clinical decision-making and early intervention in second-line treatment, which could improve patient prognosis and survival. In this real-world study, we retrospectively analyzed 82 patients with stages I to III breast cancer who had been analyzed by molecular residual disease (MRD) assay. A total of 82 tumor tissues and 224 peripheral blood samples were collected and detected by next-generation sequencing (NGS) based on a 1,021-gene panel in this study. MRD positivity was detected in 18 of 82 patients (22.0%). The hormone receptor-/human epidermal growth factor receptor 2+ (HR-/HER2+) subgroup had the highest postoperative MRD detection rate at 30.8% (4/13). The BRCA2 and SLX4 genes were significantly enriched in all patients in the MRD positive group and FGFR1 amplification was significantly enriched in the MRD negative group with HR+/HER2-. The number of single nucleotide variants (SNVs) in tissue samples of MRD-positive patients was higher than that of MRD-negative patients (11.94 vs. 8.50 SNVs/sample). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that there was a similar biological function of the tumor-mutated genes in the 2 MRD status groups. This real-world study confirmed that patient samples of primary tumor tissue with different MRD status and molecular subtypes had differential genetic features, which may be used to predict patients at high risk for recurrence.

P12.10.A Supratentorial glioblastoma with spinal metastasis: a case report with molecular profiling

Abstract Background Glioblastoma (GBM) is regarded as an aggressive brain tumor that rarely develops extracranial metastases. Despite well-investigated molecular alterations in GBM, there is a limited understanding of these associated with the metastatic potential of GBM. Material and Methods We present a case report of a 43-year-old woman with frontal GBM IDH-wildtype, who underwent gross-total resection followed by chemoradiation. Five months after surgery, the patient was diagnosed with an intraspinal GBM metastasis. Next-generation sequencing analysis of both primary and metastatic GBM tissues was performed using the Illumina TruSight Tumor 170 assay. Results The number of single nucleotide variants observed in the metastatic sample was more than 2-times higher. Mutations in TP53, PTEN, and RB1 found in the primary and metastatic tissue samples are indicative of the mesenchymal GBM subtype. Among others, in the metastatic sample, there were detected two inactivating mutations (Arg1026Ile, Trp1831Ter) in the NF1 gene, two novel NOTCH3 variants of unknown significance predicted to be damaging (Pro1505Thr, Cys1099Tyr), one novel ARID1A variant of unknown significance (Arg1046Ser), and one gene fusion of unknown significance EIF2B5-KIF5B. Conclusion Based on the literature evidence, the inactivation of NF1, NOTCH3, and ARID1A could explain, at least in part, the acquired invasiveness and metastatic potential in this particular GBM case. This work was supported by the grant MUNI/A/1408/2021 of the Masaryk University, Brno, Czech Republic, and INTER-EXCELLENCE Programme / INTER-COST project no. LTC20027 of the Ministry of Health, Czech Republic.

Targeted Next-Generation Sequencing of Thymic Epithelial Tumours Revealed Pathogenic Variants in KIT, ERBB2, KRAS, and TP53 in 30% of Thymic Carcinomas.

Simple SummaryThe biology of thymic epithelial tumours (TETs), including thymomas and thymic carcinomas, and particularly the extent of molecular dysregulation, is poorly understood. Through next-generation sequencing of 15 genes implicated in common solid tumours in 53 TETs, we found a larger number of single nucleotide variants (SNVs) in thymic carcinomas than thymomas. About 30% of thymic carcinomas had at least one somatic pathogenic gene variant in TP53, ERBB2, KIT, or KRAS, whereas variants of uncertain clinical significance in KIT, ERBB2, and FOXL2 were found exclusively in thymomas. The presence of somatic pathogenic variants was non-significantly associated with shorter disease-free survival in thymic carcinoma patients. No somatic pathogenic or likely pathogenic SNV was found in thymomas. Importantly, we also evaluated germline SNVs, adding to the number of known genetic alterations in TETs and thereby enhancing our molecular understanding of these neoplasms.A better understanding of the molecular pathogenesis of thymic epithelial tumours (TETs) could revolutionise their treatment. We evaluated thymomas and thymic carcinomas by next-generation sequencing (NGS) of somatic or germline single nucleotide variants (SNVs) in genes commonly mutated in solid tumours. In total, 19 thymomas and 34 thymic carcinomas were analysed for nonsynonymous SNVs in 15 genes by targeted NGS (reference genome: hg19/GRCh37). Ten SNVs in TP53 (G154V, R158P, L194H, R267fs, R273C, R306 *, Q317 *), ERBB2 (V773M), KIT (L576P), and KRAS (Q61L) considered somatic and pathogenic/likely pathogenic were detected in 10 of 34 (29.4%) thymic carcinomas. No somatic SNVs confirmed as pathogenic/likely pathogenic were found in thymomas. Rare SNVs of uncertain or unknown functional and clinical significance, to our knowledge not reported previously in TETs, were found in ERBB2 (S703R), KIT (I690V), and FOXL2 (P157S) in 3 of 19 (16%) thymomas. The most frequent germline SNVs were TP53 P72R (94% TETs), ERBB2 I655V (40% TETs), and KIT M541L (9% TETs). No significant difference in median disease-free survival (DFS) was found between thymic carcinoma patients with and without pathogenic SNVs (p = 0.190); however, a trend toward a longer DFS was observed in the latter (16.0 vs. 30.0 months, respectively). In summary, NGS analysis of TETs revealed several SNVs in genes related to the p53, AKT, MAPK, and K-Ras signalling pathways. Thymic carcinomas showed greater genetic dysregulation than thymomas. The germline and rare SNVs of uncertain clinical significance reported in this study add to the number of known genetic alterations in TETs, thus extending our molecular understanding of these neoplasms. Druggable KIT alterations in thymic carcinomas have potential as therapeutic targets.