Non-synonymous Variants Research Articles

Abstract 2522: Predictive value of tumor mutational burden (TMB) in patients with metastatic microsatellite-stable (MSS) colorectal cancer (CRC) given first-line oxaliplatin-based chemotherapy and immune checkpoint blockade (ICB)

Abstract Purpose: Most CRC patients harbor primarily non-immunogenic MSS tumor. The randomized METIMMOX trial (NCT03388190) examined if patients with previously untreated, unresectable abdominal metastases from MSS-CRC may achieve therapeutic efficacy from short-course oxaliplatin-based chemotherapy (FLOX) and sequential ICB (nivolumab) repeatedly. Half of study patients assigned to this experimental treatment had significantly extended progression-free survival (PFS) while the other half had shortened PFS, compared to the control-group patients given standard FLOX chemotherapy with median PFS 9.3 months. Here we explored if somatic mutations (mut) unveiled by next-generation sequencing might provide insights into responsiveness to the METIMMOX regimen. Procedures: Patients had MSS-CRC with infradiaphragmatic metastases deemed unresectable and were eligible for first-line oxaliplatin-based therapy. They were randomly assigned to the control group of FLOX (oxaliplatin, 5-fluorouracil, folinic acid) Q2W or the experimental group of alternating 2 cycles each of FLOX Q2W and nivolumab Q2W, with prespecified break periods. Radiologic response assessment was done every 8 weeks with PFS as the primary endpoint. Diagnostic tumor biopsies and baseline metastasis biopsies were sequenced with the TruSight Oncology 500 assay. TMB was calculated using only coding, non-synonymous single-nucleotide variants and insertions/deletions with variant allele frequency ≥5% and sequencing depth ≥50 ×, applying the effective panel size as the megabases (Mb) denominator. Results: Sequencing data were acquired from 20 experimental-group patients with median PFS 7.9 months (minimum 2.0, maximum 41.6). Median TMB was 5.1 mut/Mb (minimum 0.8, maximum 11.8), concordant in patient-paired primary tumor and metastasis specimens. The TMB was associated with treatment outcome; the higher TMB, the better PFS with hazard ratio 0.83 (95% confidence interval (CI) 0.70-0.98; p = 0.026, Cox regression) for a PFS event with increasing TMB. TMB cut-off as low as 5.0 mut/Mb distinguished between patient groups (p = 0.007, log-rank test) with median PFS 34.9 months (95% CI 10.6-59.2; TMB>5, n = 10) and median PFS 4.6 months (95% CI 2.7-6.5; TMB≤5, n = 10). In the TMB>5 group, 8/10 were KRAS- or BRAF-mutant cases. In the TMB≤5 group, 9/10 metastasis specimens were RAS/BRAF-wildtype cases. Conclusions: TMB >5 mut/Mb was associated with tumor KRAS or BRAF driver mutation and remarkably long PFS in first-line treatment of patients with abdominal metastases from MSS-CRC with alternating short-course oxaliplatin-based chemotherapy and ICB. Citation Format: Anne Hansen Ree, Paula A. Bousquet, Hilde L. Nilsen, Torben Lüders, Shixiong Wang, Tina Visnovska, Diana L. Bordin, Eirik Høye, Hanne M. Hamre, Christian Kersten, Eva Hofsli, Marianne G. Guren, Halfdan Sorbye, Kjersti Flatmark, Sebastian Meltzer. Predictive value of tumor mutational burden (TMB) in patients with metastatic microsatellite-stable (MSS) colorectal cancer (CRC) given first-line oxaliplatin-based chemotherapy and immune checkpoint blockade (ICB) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2522.

Abstract 7048: Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages

Abstract Recent epidemiological data suggests a rising incidence of breast angiosarcoma (AS-B) in the Western population, with over two-thirds related to irradiation or chronic lymphedema. However, unlike head and neck angiosarcoma (AS-HN), AS-B disease characteristics in Asia remains unclear. We examined clinical patterns of angiosarcoma patients (n = 176) seen in an Asian tertiary cancer centre from 1999 to 2021, and specifically investigated the molecular and immune features of AS-B in comparison to AS-HN. Data from whole genome sequencing (WGS) (AS-HN, n = 44; breast, n = 7), NanoString PanCancer IO360 gene expression profiling (AS-HN, n = 42; breast, n = 7) and 10X Visium spatial transcriptomics (AS-HN, n = 4; breast, n = 2) were analyzed. Majority of cases were AS-HN (n = 104; 59.1%), while AS-B (n = 16, all females) accounted for 9.1% of the cases. The median age at diagnosis was 43 years (range, 26 to 74). Four cases (25%) were related to prior irradiation, while 6 out of 8 cases evaluated were positive for human herpesvirus-7 on immunohistochemistry. Although all cases were non-metastatic at diagnosis and were treated with curative-intent surgery, 6 patients (37.5%) experienced rapid relapse within a median of 7.8 months. Based on WGS, 4 of the 7 AS-B had non-synonymous somatic variants in 48 genes (range, 2 to 28 per case). Among the notable variants found were in kinases (KDR, JAK2, FLT4, ATR, EPHB1 and PRKACA). Other variants were found in cancer-related genes (e.g. ATRX, FOXO1, SMAD3, PTCH1, NOTCH4, HIF1A and SETD1A). These genes were functionally annotated by performing over representation analysis (www.webgestalt.org) on publicly available databases (e.g. Gene Ontology and KEGG). They were enriched in cancer-related pathways such as regulation of cell differentiation, VEGFR and receptor tyrosine kinase signaling pathways. By NanoString profiling, compared to AS-HN, AS-B was enriched for angiogenesis, notch signaling and metastasis-associated matrix remodeling pathways. Conversely, AS-B was depleted of lymphoid compartment, immune cell adhesion and migration expression signatures. Based on the gene expression signatures, AS-B was enriched for macrophages, CD8+ T cells and was depleted for T-reg cells. Furthermore, spatial transcriptomics showed that compared to AS-HN, tumor-infiltrating leukocytes (TILs) in AS-B were enriched for macrophage and T-cells but were depleted for B-cells and NK cells. The location of TILs could provide information on the feasibility of immunotherapy in angiosarcoma. In conclusion, we observed a convergence of both mutational and expression signatures on angiogenesis signaling pathways in our AS-B cases. The spatial transciptomic data revealed that AS-B was enriched for tumor-infiltrating macrophages and T-cells which might have implications on the role of immunotherapy. Citation Format: Tun Kiat Ko, Zexi Guo, Bavani Kannan, Boon Yee Lim, Jing Yi Lee, Abner Herbert Lim, Zhimei Li, Elizabeth Chun Yong Lee, Cedric Chuan-Young Ng, Bin Tean Teh, Jason Yongsheng Chan. Multiomics characterization of breast angiosarcoma from an Asian cohort reveals enrichment for angiogenesis signaling pathway and tumor-infiltrating macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7048.

Abstract 1747: The mutational landscape and its longitudinal dynamics in relapsed and refractory Hodgkin lymphoma

Abstract Introduction: In classical Hodgkin-lymphoma (cHL), only a few cases recur, and only a limited fraction of patients is primary-refractory to standard-polychemotherapy. Underlying genomic features of unfavorable clinical courses remain sparsely characterized. Here, we investigated the genomic characteristics of primary-refractory/relapsed cHL in contrast with long-term-responders (LTR). Methods: Therefore, ultra-deep next-generation panel-sequencing was performed on a total of 59 samples (20 LTR, 26 relapsed (rHL: 11 initial-diagnosis, 15 relapse) and 13 primary-refractory (prHL: 8 initial-diagnosis, 5 progression) from 44 cHL-patients applying a hybrid-capture approach. We compared samples associated with distinct disease courses concerning their oncogenic drivers, mutational signatures, and perturbed pathways. Results: Compared to LTRs, mutations in genes such as PMS2, PDGFRB, KAT6A, EPHB1, and HGF were detected more frequently in prHL/rHL. Additionally, we observed that in rHL or prHL, BARD1-mutations occur, whereas ETV1, NF1, and MET-mutations were eliminated through clonal selection. A significant enrichment of non-synonymous variants was detected in prHL compared to LTRs and a significant selection process in favor of NOTCH-pathway mutations driving rHL or prHL was observed. Conclusion: This study delineates distinct mutational signatures between LTR and rHL/prHL, whilst illustrating longitudinal dynamics in mutational profiles using paired samples. Further, several exploitable therapeutic vulnerabilities for rHL and prHL were identified. Citation Format: Hanno Witte, Axel Künstner, Thomas Hahn, Veronica Bernard, Stephanie Stoelting, Kathrin Kusch, Kumar Nagarathinam, Nikolas von Bubnoff, Cyrus Khandanpour, Arthur Bauer, Michael Grunert, Annette Arndt, Konrad Steinestel, Hartmut Merz, Hauke Busch, Alfred Feller, Niklas Gebauer. The mutational landscape and its longitudinal dynamics in relapsed and refractory Hodgkin lymphoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1747.

Identification of a rare MET variant in three siblings with extramammary Paget disease.

Extramammary Paget disease (EMPD) is an intraepithelial adenocarcinoma that primarily affects the genital and axillary areas in older individuals. A limited number of paired patients with familial EMPD (i.e. parent-offspring, siblings) have been reported but the genetics have not yet been adequately studied. We report, to the best of our knowledge, the first familial cases of patients with EMPD involving three affected siblings. The tumour-only multigene panel testing using surgical specimens revealed a heterozygous c.2997A>C (p.Glu999Asp) nonsynonymous variant in the proto-oncogene MET (NM_000245.4) in the three affected siblings. The germline multigene panel testing using peripheral blood lymphocytes revealed the same missense MET variant in all five family members who were tested, including two asymptomatic offspring (51 and 37 years of age). The MET variant we identified could be involved in EMPD carcinogenesis. Further genomic analyses of patients with familial EMPD are warranted to validate the pathogenic relevance of MET variants in EMPD development.

Development of New PCR Protocols to Detect Genetic Diversity in the Metronidazole Metabolism Genes in Susceptible and Refractory Clinical Samples of Giardia duodenalis.

Investigating the genetic variation in thioredoxin reductase (TrxR) and nitroreductase (NR) genes in both treatment-resistant and -sensitive Giardia duodenalis isolates can provide valuable information in identifying potential markers of resistance to metronidazole. The rapid increase in metronidazole treatment failures suggests the presence of genetic resistance mechanisms. By analyzing these genes, researchers can gain insights into the efficacy of metronidazole against G. duodenalis and potentially develop alternative treatment strategies. In this regard, four G. duodenalis isolates (two clinically sensitive and two clinically resistant to metronidazole) were collected from various hospitals of Shiraz, southwestern Iran. Parasitological methods including sucrose flotation and microscopy were employed for the primary confirmation of G. duodenalis cysts in stool samples. Microscopy-positive samples were approved by SSU-PCR amplification of the parasite DNA. All four positive G. duodenalis specimens at SSU-PCR were afterward analyzed utilizing designed primers based on important metronidazole metabolism genes including TrxR, NR1, and NR2. Unlike TrxR gene, the results of NR1 and NR2 genes showed that there are non-synonymous variations between sequences of treatment-sensitive and -resistant samples compared to reference sequences. Furthermore, the outcomes of molecular docking revealed that there is an interaction between the protein sequence and spatial shape of treatment-resistant samples and metronidazole in the position of serine amino acid based on the NR1 gene. This issue can be one of the possible factors involved in the resistance of Giardia parasites to metronidazole. To reach more accurate results, a large sample size along with simulation and advanced molecular dynamics investigations are needed.

Variación molecular de genes asociados a lipoxigenasas en grano de variedades de soya comercial mexicana

Lipoxygenase enzymes encoded by the Lox1, Lox2 and Lox3 genes play a crucial role in soybean grain, particularly in the development of off-flavors. Understanding molecular variation within Lox genes is essential for the improvement of soybean organoleptic traits. This study investigated the genetic variation in the internal regions of the Lox1, Lox2, and Lox3 genes in mature grain of commercially grown soybean cultivars in Mexico. Genomic DNA from a diverse panel of Mexican soybean cultivars was analyzed using resequencing techniques and in-silico analysis. Single nucleotide polymorphisms (SNP) within the Lox1, Lox2, and Lox3 genes were identified and characterized. The findings indicated that Lox3 gene displayed lower genetic variability compared to Lox1 and Lox2 genes, specifically, was identified a total of 26 SNPs in the Lox1 gene, 11 SNPs in the Lox2 gene, and 5 SNPs in the Lox3 gene among the examined cultivars. A non-synonymous SNP variant of the C/C genotype located in exon 6 of the Lox2 gene was associated with a destabilizing effect on the lipoxygenase 2 enzyme in the Guayparime S-10 and Huasteca 300 cultivars. These findings provide insights into the molecular variation of lipoxygenase-associated genes in Mexican soybean cultivars.

The effect of marker types and density on genomic prediction and GWAS of key performance traits in tetraploid potato.

Genomic prediction and genome-wide association studies are becoming widely employed in potato key performance trait QTL identifications and to support potato breeding using genomic selection. Elite cultivars are tetraploid and highly heterozygous but also share many common ancestors and generation-spanning inbreeding events, resulting from the clonal propagation of potatoes through seed potatoes. Consequentially, many SNP markers are not in a 1:1 relationship with a single allele variant but shared over several alleles that might exert varying effects on a given trait. The impact of such redundant "diluted" predictors on the statistical models underpinning genome-wide association studies (GWAS) and genomic prediction has scarcely been evaluated despite the potential impact on model accuracy and performance. We evaluated the impact of marker location, marker type, and marker density on the genomic prediction and GWAS of five key performance traits in tetraploid potato (chipping quality, dry matter content, length/width ratio, senescence, and yield). A 762-offspring panel of a diallel cross of 18 elite cultivars was genotyped by sequencing, and markers were annotated according to a reference genome. Genomic prediction models (GBLUP) were trained on four marker subsets [non-synonymous (29,553 SNPs), synonymous (31,229), non-coding (32,388), and a combination], and robustness to marker reduction was investigated. Single-marker regression GWAS was performed for each trait and marker subset. The best cross-validated prediction correlation coefficients of 0.54, 0.75, 0.49, 0.35, and 0.28 were obtained for chipping quality, dry matter content, length/width ratio, senescence, and yield, respectively. The trait prediction abilities were similar across all marker types, with only non-synonymous variants improving yield predictive ability by 16%. Marker reduction response did not depend on marker type but rather on trait. Traits with high predictive abilities, e.g., dry matter content, reached a plateau using fewer markers than traits with intermediate-low correlations, such as yield. The predictions were unbiased across all traits, marker types, and all marker densities >100 SNPs. Our results suggest that using non-synonymous variants does not enhance the performance of genomic prediction of most traits. The major known QTLs were identified by GWAS and were reproducible across exonic and whole-genome variant sets for dry matter content, length/width ratio, and senescence. In contrast, minor QTL detection was marker type dependent.

Two-stage association study of mitochondrial DNA variants in allergic rhinitis

BackgroundCorrelations between mitochondrial DNA (mtDNA) and allergic rhinitis (AR) have not been reported before. This study aimed to better understand the mitochondrial genome profile with AR and to investigate the associations between AR in China and the mitochondrial genome at a single variant and gene level.MethodsMitochondrial sequencing was conducted on a total of 134 unrelated individual subjects (68 patients with AR, 66 healthy controls) at discovery stage. Heteroplasmy was analyzed using the Mann-Whitney U test. Sequence kernel association tests (SKAT) were conducted to study the association between mitochondrial genes and AR. Single-variant analysis was performed using logistic regression analysis and further validated in 120 subjects (69 patients with AR, 51 healthy controls). Candidate genes were further explored based on differences in mRNA and protein abundance in nasal mucosal tissue.ResultsIn the discovery stage, 886 variants, including 836 SNV and 50 indels, were identified with mitochondrial sequencing. No statistically significant differences were identified for the mitochondrial heteroplasmy or SKAT analysis between these two groups after applying a Boferroni correction. One nonsynonymous variants, rs3135028 (MT8584.G/A) in ATP6, was related to a reduced risk of AR in both the discovery and validation cohorts. Furthermore, mRNA levels of MT-ATP6 in nasal mucosal tissue were significantly lower in AR individuals than in controls (P < 0.05).ConclusionsIn a two-stage analysis of associations between AR and mtDNA variations, mitochondrial gene maps of Chinese patients with AR indicated that the ATP6 gene was probably associated with AR at the single-variant level.

Temporal dynamics of gut microbiomes in non-industrialized urban Amazonia.

Increasing levels of industrialization have been associated with changes in gut microbiome structure and loss of features thought to be crucial for maintaining gut ecological balance. The stability of gut microbial communities over time within individuals seems to be largely affected by these changes but has been overlooked among transitioning populations from low- to middle-income countries. Here, we used metagenomic sequencing to characterize the temporal dynamics in gut microbiomes of 24 individuals living an urban non-industrialized lifestyle in the Brazilian Amazon. We further contextualized our data with 165 matching longitudinal samples from an urban industrialized and a rural non-industrialized population. We show that gut microbiome composition and diversity have greater variability over time among non-industrialized individuals when compared to industrialized counterparts and that taxa may present diverse temporal dynamics across human populations. Enterotype classifications show that community types are generally stable over time despite shifts in microbiome structure. Furthermore, by tracking genomes over time, we show that levels of bacterial population replacements are more frequent among Amazonian individuals and that non-synonymous variants accumulate in genes associated with degradation of host dietary polysaccharides. Taken together, our results suggest that the stability of gut microbiomes is influenced by levels of industrialization and that tracking microbial population dynamics is important to understand how the microbiome will adapt to these transitions.IMPORTANCEThe transition from a rural or non-industrialized lifestyle to urbanization and industrialization has been linked to changes in the structure and function of the human gut microbiome. Understanding how the gut microbiomes changes over time is crucial to define healthy states and to grasp how the gut microbiome interacts with the host environment. Here, we investigate the temporal dynamics of gut microbiomes from an urban and non-industrialized population in the Amazon, as well as metagenomic data sets from urban United States and rural Tanzania. We showed that healthy non-industrialized microbiomes experience greater compositional shifts over time compared to industrialized individuals. Furthermore, bacterial strain populations are more frequently replaced in non-industrialized microbiomes, and most non-synonymous mutations accumulate in genes associated with the degradation of host dietary components. This indicates that microbiome stability is affected by transitions to industrialization, and that strain tracking can elucidate the ecological dynamics behind such transitions.

A comparative genomic approach to decipher the mutations associated with Nipah viral human isolates from southeast Asia.

Multiple outbreaks over two decades and a high mortality rate have emphasized the Nipah virus (NiV) as a priority research area. The study focuses on identifying the mutational landscape in sequences from NiV human isolates from different geographical regions. Thirty-seven NiV genomes of human samples from Malaysia, Bangladesh, and India were subjected to phylogeny and metagenomic analysis to decipher the genome variability using MEGA11 software and the meta-CATS web server. Using the Single-Likelihood Ancestor Counting method, the synonymous and nonsynonymous mutations among NiV genes were identified. Further, the nonsynonymous variations were used to identify mutations in all the NiV proteins. The NiV isolates were categorized into NiV-M, NiV-B, and NiV-I clades based on phylogenetic analysis. Metagenomic analysis revealed 1636 variations in the noncoding and coding regions of the genomes of the three clades of NiV. Further analysis of nonsynonymous mutations showed the phosphoprotein to be highly mutating, whereas the matrix protein was stable. Deciphering the mutation pattern using a comparative genomics approach for human isolates provided valuable insight into the stability of NiV proteins which can be further used for understanding variations in host-pathogen interaction and developing effective therapeutic measures.

Rare and common coding variants in lipid metabolism-related genes and their association with coronary artery disease

BackgroundCoronary artery disease (CAD) is a complex disease that is influenced by environmental and genetic factors. In this study, we aimed to investigate the relationship between coding variants in lipid metabolism-related genes and CAD in a Chinese Han population.MethodsA total of 252 individuals were recruited for this study, including 120 CAD patients and 132 healthy control individuals. Rare and common coding variants in 12 lipid metabolism-related genes (ANGPTL3, ANGPTL4, APOA1, APOA5, APOC1, APOC3, CETP, LDLR, LIPC, LPL, PCSK9 and SCARB1) were detected via next-generation sequencing (NGS)-based targeted sequencing. Associations between common variants and CAD were evaluated by Fisher’s exact test. A gene-based association test of rare variants was performed by the sequence kernel association test-optimal (SKAT-O test).ResultsWe found 51 rare variants and 17 common variants in this study. One common missense variant, LIPC rs6083, was significantly associated with CAD after Bonferroni correction (OR = 0.47, 95% CI = 0.29–0.76, p = 1.9 × 10− 3). Thirty-three nonsynonymous rare variants were identified, including two novel variants located in the ANGPTL4 (p.Gly47Glu) and SCARB1 (p.Leu233Phe) genes. We did not find a significant association between rare variants and CAD via gene-based analysis via the SKAT-O test.ConclusionsTargeted sequencing is a powerful tool for identifying rare and common variants in CAD. The common missense variant LIPC rs6083 confers protection against CAD. The clinical relevance of rare variants in CAD aetiology needs to be investigated in larger sample sizes in the future.

Children born after assisted reproduction more commonly carry a mitochondrial genotype associating with low birthweight

Children conceived through assisted reproductive technologies (ART) have an elevated risk of lower birthweight, yet the underlying cause remains unclear. Our study explores mitochondrial DNA (mtDNA) variants as contributors to birthweight differences by impacting mitochondrial function during prenatal development. We deep-sequenced the mtDNA of 451 ART and spontaneously conceived (SC) individuals, 157 mother-child pairs and 113 individual oocytes from either natural menstrual cycles or after ovarian stimulation (OS) and find that ART individuals carried a different mtDNA genotype than SC individuals, with more de novo non-synonymous variants. These variants, along with rRNA variants, correlate with lower birthweight percentiles, independent of conception mode. Their higher occurrence in ART individuals stems from de novo mutagenesis associated with maternal aging and OS-induced oocyte cohort size. Future research will establish the long-term health consequences of these changes and how these findings will impact the clinical practice and patient counselling in the future.

(304) Effects of Wireless Radiation Exposure on Genetic Variants in Human Sperm: A Preliminary Prospective Study

Abstract Introduction In the present era, the extensive use of modern cell phones has raised the health risks associated with the radiofrequency-electromagnetic radiation (RF-EMR) they emit, which the human body can absorb. RF-EMR exposure can potentially harm the process of spermatogenesis by increasing oxidative stress, elevating mitochondrial reactive oxygen species (ROS), and triggering the formation of free radicals that leads to sperm DNA damage. Our previous research revealed decreased sperm motility and viability in semen samples exposed to RF-EMR through WiFi calling. Objective To investigate the effects of WiFi radiation on genetic variants in human sperm using current-generation smartphones. Methods Six healthy, normozoospermic men aged 25-35 provided semen samples for this study. A current generation smartphone was used in WiFi calling mode, specifically through Whatsapp voice call, to serve as the source of RF-EMR. The call duration was six hours. Semen samples were analyzed based on the 2010 WHO guidelines before and after the radiation exposure. The genomics facility at our university extracted DNA from semen samples of each participant before and after RF-EMR exposure to perform whole exome sequencing with alignment to the reference genome. After sequencing, we compared genetic variants for each participant before and after RF-EMR exposure. Results After six hours of exposure, a significant decrease in sperm progressive motility was observed (p&amp;lt;0.05). Following exposure, the median number of exonic, nonsynonymous variants absent before exposure increased to 4 (IQR 3). Across the four successfully sequenced samples, we found 20 new, exonic, nonsynonymous variants after RF-EMR exposure. Significantly, male reproductive tissues expressed 12 variants, five associated with ciliary function and two related to mitochondrial function (see Table 1). Gene Chromosome Variant Location Function SPDYE11 Chr7 A &amp;gt; C Spermatids • Acetylcholine receptor activity • Cell cycle regulation SPDYE17 Chr7 A &amp;gt; C Spermatids • Protein kinase activity • Cell cycle regulation PRB2 Chr12 T &amp;gt; G salivary gland • Cell cycle regulation GGT2 Chr22 G &amp;gt; A Spermatids • Cell cycle regulation RGPD3 Chr2 C &amp;gt; T Spermatids • Ciliary function EPPK1 Chr8 G &amp;gt; A Spermatids, spermatocytes • Epithelial structure GOLGA6L4 Chr15 G &amp;gt; A Spermatids, spermatocytes • Ciliary function TCP11X2 ChrX T &amp;gt; C Spermatids • Acrosome reaction • Cilia function • Spermatid development HRNR Chr1 A &amp;gt; G Mitochondria • Metal ion binding POUF1 Chr13 &amp;gt; GCC Mitochondria • Structure &amp; function of spermatozoa NPIPB13 Chr16 T &amp;gt; A Spermatids • Ciliary function • Sperm motility NBPF11 Chr1 A &amp;gt; C Spermatids, Sertoli cells, Leydig cells • Cell cycle regulation • Microtubule development ANKRD20A1 Chr9 T &amp;gt; A Spermatocytes • Ciliary function Table 1. Candidate genes with variants after six hours of phone radiation exposure Conclusions Based on our pilot study, we observed a decrease in sperm progressive motility and an increase in potentially related genetic variants associated with sperm motility following six hours of exposure to RF-EMR. These preliminary results highlight the need for further research to investigate the effects of RF-EMR exposure on semen parameters and to determine the clinical significance of these genetic variants. Disclosure No.

Exploring the Role of Non-synonymous and Deleterious Variants Identified in Colorectal Cancer: A Multi-dimensional Computational Scrutiny of Exomes.

Colorectal cancers are the world's third most commonly diagnosed type of cancer. Currently, there are several diagnostic and treatment options to combat it. However, a delay in detection of the disease is life-threatening. Additionally, a thorough analysis of the exomes of cancers reveals potential variation data that can be used for early disease prognosis. By utilizing a comprehensive computational investigation, the present study aimed to reveal mutations that could potentially predispose to colorectal cancer. Ten colorectal cancer exomes were retrieved. Quality control assessments were performed using FastQC and MultiQC, gapped alignment to the human reference genome (hg19) using Bowtie2 and calling the germline variants using Haplotype caller in the GATK pipeline. The variants were filtered and annotated using SIFT and PolyPhen2 successfully categorized the mutations into synonymous, non-synonymous, start loss and stop gain mutations as well as marked them as possibly damaging, probably damaging and benign. This mutational profile helped in shortlisting frequently occurring mutations and associated genes, for which the downstream multi-dimensional expression analyses were carried out. Our work involved prioritizing the non-synonymous, deleterious SNPs since these polymorphisms bring about a functional alteration to the phenotype. The top variations associated with their genes with the highest frequency of occurrence included LGALS8, CTSB, RAD17, CPNE1, OPRM1, SEMA4D, MUC4, PDE4DIP, ELN and ADRA1A. An in-depth multi-dimensional downstream analysis of all these genes in terms of gene expression profiling and analysis and differential gene expression with regard to various cancer types revealed CTSB and CPNE1 as highly expressed and overregulated genes in colorectal cancer. Our work provides insights into the various alterations that might possibly lead to colorectal cancer and suggests the possibility of utilizing the most important genes identified for wet-lab experimentation.

Mutation in BrFLS encoding flavonol synthase induced anthocyanin accumulation in Chinese cabbage.

BrFLS mutation promoted anthocyanin accumulation in Chinese cabbage, which was verified in four allelic mutants. Chinese cabbage is a major vegetable crop in Eastern Asia. Anthocyanin-rich vibrantly colored varieties are increasingly favored by consumers for their higher nutritional and aesthetic value compared to the typical green varieties of Chinese cabbage. Herein, we identified an anthocyanin accumulation mutant aam1 from a mutant library of EMS-mutagenized Chinese cabbage DH line 'FT', which appeared partial purple on leaves, bolting stems and floral buds. This anthocyanin accumulation trait was genetically controlled by a recessive nuclear gene, and through MutMap mapping and KASP genotyping, BraA10g030950.3C was identified as the candidate causal gene with a G202 to A202 non-synonymous SNP variation in exon 1. Three additional mutants allelic to aam1 were obtained via screening of similar-phenotype mutants from the mutant library, namely aam2/3/4, where the causal SNPs reside in the same gene as aam1, corroborating that the mutation of BraA10g030950.3C caused anthocyanin accumulation. BraA10g030950.3C encodes a flavonol synthase that catalyzes dihydroflavonols substrate into flavonols and is homologous to Arabidopsis FLS1 (AT5G08640), named BrFLS. Compared to wildtype, the expression level of BrFLS was significantly reduced in the mutants, while BrDFR, which is involved in the anthocyanin biosynthesis and competes with FLS for the common substrate dihydroflavonols, was increased. The flavonol synthase activity decreased, and dihydroflavonol 4-reductase activity was elevated. Differentially accumulated flavonoid metabolites were detected between wildtype and aam1, which were enriched primarily in flavonol and anthocyanin pathways. Our results revealed that mutations in the BrFLS gene could contribute to anthocyanin accumulation and provide a new target for Chinese cabbage color modification.

Canine coat color E locus updates: Identification of a new MC1R variant causing 'sable' coat color in English Cocker Spaniels and a proposed update to the E locus dominance hierarchy.

The coat color phenotype 'sable' occurs in the English Cocker Spaniel dog breed. It closely resembles other canine color patterns known as domino/grizzle/pied (eA allele) and grizzle/domino (eG allele) determined by variants in the melanocortin 1 receptor gene (MC1R; 'extension' or E locus), a key multi-allele regulator of coat color. We examined genetic variation in MC1R, and found one new non-synonymous variant, c.250G>A (p.(Asp84Asn)), consistently associated with the English Cocker Spaniel 'sable' phenotype. We propose calling this newly identified allele eH and further show that the eA , eH and eG (previously known as EG ) alleles associate with similar phenotypes in dogs impacting genotypes regulated by beta-defensin 103 gene (CBD103; K locus) and agouti signaling protein gene (ASIP; A locus) in the absence of the EM and E alleles. This suggests that all three alleles are putative reduced-function variants of the MC1R gene. We propose the revised and updated E locus dominance hierarchy to be EM > E > eA /eH /eG > e1-3 .

Transcriptomic insights into shared responses to Fusarium crown rot infection and drought stresses in bread wheat (Triticum aestivum L.)

Key messageShared changes in transcriptomes caused by Fusarium crown rot infection and drought stress were investigated based on a single pair of near-isogenic lines developed for a major locus conferring tolerance to both stresses.Fusarium crown rot (FCR) is a devastating disease in many areas of cereal production worldwide. It is well-known that drought stress enhances FCR severity but possible molecular relationship between these two stresses remains unclear. To investigate their relationships, we generated several pairs of near isogenic lines (NILs) targeting a locus conferring FCR resistance on chromosome 2D in bread wheat. One pair of these NILs showing significant differences between the two isolines for both FCR resistance and drought tolerance was used to investigate transcriptomic changes in responsive to these two stresses. Our results showed that the two isolines likely deployed different strategies in dealing with the stresses, and significant differences in expressed gene networks exist between the two time points of drought stresses evaluated in this study. Nevertheless, results from analysing Gene Ontology terms and transcription factors revealed that similar regulatory frameworks were activated in coping with these two stresses. Based on the position of the targeted locus, changes in expression following FCR infection and drought stresses, and the presence of non-synonymous variants between the two isolines, several candidate genes conferring resistance or tolerance to these two types of stresses were identified. The NILs generated, the large number of DEGs with single-nucleotide polymorphisms detected between the two isolines, and the candidate genes identified would be invaluable in fine mapping and cloning the gene(s) underlying the targeted locus.

Compound heterozygous WNT10A missense variations exacerbated the tooth agenesis caused by hypohidrotic ectodermal dysplasia

BackgroundThe aim of this study was to analyse the differences in the phenotypes of missing teeth between a pair of brothers with hypohidrotic ectodermal dysplasia (HED) and to investigate the underlying mechanism by comparing the mutated gene loci between the brothers with whole-exome sequencing.MethodsThe clinical data of the patients and their mother were collected, and genomic DNA was extracted from peripheral blood samples. By Whole-exome sequencing filtered for a minor allele frequency (MAF) ≤0.05 non-synonymous single-nucleotide variations and insertions/deletions variations in genes previously associated with tooth agenesis, and variations considered as potentially pathogenic were assessed by SIFT, Polyphen-2, CADD and ACMG. Sanger sequencing was performed to detect gene variations. The secondary and tertiary structures of the mutated proteins were predicted by PsiPred 4.0 and AlphaFold 2.ResultsBoth brothers were clinically diagnosed with HED, but the younger brother had more teeth than the elder brother. An EDA variation (c.878 T > G) was identified in both brothers. Additionally, compound heterozygous variations of WNT10A (c.511C > T and c.637G > A) were identified in the elder brother. Digenic variations in EDA (c.878 T > G) and WNT10A (c.511C > T and c.637G > A) in the same patient have not been reported previously. The secondary structure of the variant WNT10A protein showed changes in the number and position of α-helices and β-folds compared to the wild-type protein. The tertiary structure of the WNT10A variant and molecular simulation docking showed that the site and direction where WNT10A binds to FZD5 was changed.ConclusionsCompound heterozygous WNT10A missense variations may exacerbate the number of missing teeth in HED caused by EDA variation.

Integrated rules classifier for predicting pathogenic non-synonymous single nucleotide variants in human

The most prevalent kind of genetic variants in humans are non-synonymous single nucleotide variants (nsSNVs). Several prediction tools have been launched to forecast the effect of amino acid substitutes on human protein function. These tools sort variants as pathogenic or neutral. We developed an Integrated Rules Classifier (Integration Score through JRip “ISTJRip”), which integrates the four individual tools that are publicly available; iFish, Mutation Assessor, FATHMM, and SIFT-based on the JRip machine learning technique. Additionally, we compared the ISTJRip approach with the other three created integration classifiers; Integration Score through J48 “ISTJ48”, Integration Score through RF “ISTRF”, and Integration Score through SVM “ISTSVM” using a VaribenchSelectedPure dataset character from the standard dataset “Varibench”. The proposed integrated rules classifier “ISTJRip” and the other three integration classifiers, ISTJ48, ISTRF, and ISTSVM register 92.41 %, 92.26 %, 91.70 %, and 90.62 % ACC on VaribenchSelectedPure, respectively. Finally, we demonstrated that the integrated rules classifier outperforms other integration classifiers and highlights the benefits of JRip machine learning technique in the integration process for multiple tools.

Exploratory analysis of biomarkers of response to durvalumab in advanced HER2-negative oesophago-gastric adenocarcinoma within a phase 2 clinical trial.

384 Background: Advanced oesophago-gastric cancers have showcased varied responses to immunotherapy based on biomarkers such as tumour mutational burden, microsatellite instability, and PD-L1 expression. However, these markers may not fully capture tumour heterogeneity and evolutionary dynamics. Hence, understanding the intricate relationship between immune selection, specifically immune dN/dS,1 and tumour evolution is vital for devising personalized therapeutic strategies. Methods: In our phase 2 open label, multicentre, randomised PLATFORM trial (NCT02678182), patients received maintenance durvalumab post first-line chemotherapy. A survival advantage was not seen with maintenance durvalumab compared to surveillance and exploratory analysis of PD-L1 expression was not associated with improved survival outcomes.2 However, a subset of patients treated with durvalumab experienced durable responses and some experienced incremental radiological responses. A pilot substudy on a selected cohort (n=24) of these patients was designed to explore the potential of immunogenomics evolutionary-based metrics, notably dN/dS, as predictive biomarkers. DNA and RNA from FFPE baseline tumour samples underwent analysis, examining genomic factors including tumour mutational burden, indel signatures, PDL1 status, and immune evasion mechanisms. Furthermore, the evolutionary metric, immune dN/dS, which has implications in tumour immunoediting and its response to immunotherapies, was scrutinized. Results: A dominant indel mutational signature emerged from the analysis. The majority of patients displayed a significantly higher number of indel variants, compared to non-synonymous and synonymous single-nucleotide variants. A recurring observation was dN/dS values consistently above 1, indicating positive selection for non-synonymous somatic variants. Additionally, hypermutation in driver genes, notably TP53 and XIRP2, was detected. One patient, with a unique dN/dS &lt; 1, showcased survival beyond four years without liver metastases. Immune dN/dS &gt; 1 and no evidence of liver metastases displayed the best prognosis after treatment in contrast with those that have immune dN/dS &lt; 1 with or without liver metastases. Conclusions: Immune dN/dS emerges as a promising tool to decode tumour evolutionary mechanisms, providing profound insights into the dynamics of tumour evolution in advanced HER2 negative oesophago-gastric cancer patients receiving durvalumab immunotherapy. This pilot study underscores the potential of this novel immunogenomic approach in improving patient stratification and tailoring therapeutic strategies in cancer immunotherapy. 1. Zapata et al, Nature Genetics, 2023. 2. Fong et al, J Clin Oncol, 2021. Clinical trial information: NCT02678182 .