ALLELIC VARIATIONS OF SOS, HVNHX AND SDO GENES IN SOME BARLEY (Hordeum Vulgare L.) GENOTYPES

This study has been carried out at the Biotechnology Lab., Department of Field Crops, Faculty of Agriculture, Damascus University during the growing season 2019 - 2020, in order to detect the variations of SOS, HVNHX and SDO genes in different barley genotypes. Clear variations in the SOS, HVNHX and SDO genes, which are responsible for salinity tolerance were found among the investigated genotypes. It has been found that the variation in the amplicon size between loci per gene was very high in some cases, while there was a high degree of symmetry in other cases, and could be easily distinguished on 2% Agarose gel. The PCR results for the SOS genes (SOS1, SOS2, SOS3), HVNHX genes (HVNHX1, HVNHX2, HVNHX3) and SDO genes (Cu/Zn SODII, Cu/Zn SODI, CAT, GRI , APXIII) have shown only one morphological pattern in most of the studied genotypes, while revealed two patterns for the SOS3 gene, but the rest of genes (HVNHX1, HVNHX2, HVNHX3 Cu/Zn SODI, CAT) exhibited only one morphological pattern. The SOS3 was superior in the number of polymorphic patterns, as the number of total patterns was 14 in all the studied genotypes, but the Cu/Zn SODI showed the least number of polymorphic patterns with only 1 pattern, while the largest number (7 patterns) was detected in the genotype (H9), but the two genotypes Fourat9 and Fourat7 showed only one polymorphic pattern.

Molecular Profiling Feasibility on Cell-Free Tumoral DNA in Relapse/Refractory (R/R) Multiple Myeloma (MM) Patients Screened for Phase I Trials

Background: Despite recent advances in the treatment of multiple myeloma (MM) patients, cure remains rare. MM is in an era of intense clinical research and the molecular abnormalities landscape of refractory and relapsed (R/R) patients is of major interest for drug development. Furthermore, innovative molecular-oriented treatments for these R/R patients could be oriented by mutational characterization of myeloma plasma cells (PC). At relapse but also for clonal minimal residual disease monitoring on therapy, bone marrow aspiration is an invasive procedure that can give limited information since PC infiltration is heterogeneous and can be modest. Mutational profiling on circulating cell-free tumoral DNA (ctDNA) could be a simple and appropriate alternative. Method: We compared molecular landscape in myeloma PC versus in ctDNA in a cohort of 45 R/R MM patients screened at Gustave Roussy for a salvage therapy, most of them for a Phase I trial in the DITEP department, with a median age at time of molecular analysis of 69 years. All patients had received ≥ 1 prior lines of myeloma therapy (median: 2, range: 1-8), 33/45 (73%) had previously undergone autologous stem cell transplant and 14/45 (31%) had received prior daratumumab; 6/45 (13%) were double-refractory (to at least a proteasome inhibitor (PI) and an immunomodulatory imide drug (IMiD), 4/45 (9%) were triple-refractory and 1/45 (2%) was penta-refractory. Four patients (9%) had prior daratumumab and were also double-refractory. Paired samples, as well as normal sorted CD3 + T cells were sequenced using an Ion Torrent custom panel covering 30 myeloma-related genes previously reported as the most frequent mutated ones. The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol. Results: One hundred and two variants were found in magnetic-sorted CD138 + myeloma PC, and 99 variants were detected in ctDNA; more than half of the variants detected in myeloma PC were also found in ctDNA (55/102, 54%). Variant allelic frequencies (VAF) in PC and in ctDNA were significantly correlated (p<0.001). Mean VAF was 25% in myeloma PC (median 19%, range: 0.40-99%) and 5.4% in ctDNA (median 0.33%, range: 0-50%) considering the 102 mutations found in myeloma PC. KRAS, NRAS, FAM46C, DIS3 and TP53 were the most frequently mutated genes (i.e. >10% of R/R MM). Considering these five key driver genes, the kappa coefficient of concordance per gene was medium/good between both samples, as defined by the Landis-Koch scale. The mean and median sensitivity of ctDNA detection per gene was 55% and 58% respectively (range: 38-67), and specificity 94% and 97% (range: 80-100); positive predictive value of TP53 mutations detection was poor as theses mutations were more frequently detected in ctDNA (12/45, 27%) than in myeloma PC (6/45, 13%). At the patient level, the similarity between myeloma PC and ctDNA (level defined as the ratio between SNV number in PC and SNV number in ctDNA) was greater than a threshold of 80% in 20/39 (51%) cases (median level: 100%). Importantly, key driver gene mutations were reported in ctDNA for 13/28 (48%) patients without cytological evidence of infiltrated plasmocytosis (less than 10% PC) in the bone marrow aspiration. Conclusions: ctDNA profiling may complete molecular description of R/R MM patients thanks to a less-invasive procedure, allowing to fully characterize mutational profile prior to molecular-oriented treatment decision. ctDNA can give information on the clonal architecture in patients without bone marrow infiltration even after CD138 + cells magnetic-sorted isolation. Disclosures Michot: BMS: Consultancy, Honoraria; GSK: Consultancy, Honoraria. Lazarovici: Mundipharma: Other: Travel grant. Ribrag: Infinity Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Astex Pharmaceuticals: Research Funding; Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Argen-X: Research Funding; GSK: Research Funding; Epizyme: Honoraria, Research Funding; MSD Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; PharmaMar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees.

High-Plex Multiomic Analysis in FFPE Tissue at Single-Cellular and Subcellular Resolution by Spatial Molecular Imaging

Spatial Molecular Imager (SMI) is an automated microscope imaging system with microfluidic reagent cycling, for high-plex, spatial in-situ detection of multiomic targets (RNA and protein) on FFPE and other intact samples with subcellular resolution. The key attributes of the CosMx™ SMI platform (NanoString®, Seattle, WA) include: 1) high-plex and high-sensitivity imaging chemistry that works for both RNA and protein detection, 2) three-dimensional subcellular-resolution image analysis with a target localization accuracy of ~50 nm in the XY plane, 3) large Hamming-distance encoding scheme with low error rate (0.0092 false calls per cell per gene) and low background (~ 0.04 counts per cell per gene), 4) high-throughput (up to 1 million cells per sample, four samples per run), 5) antibody-based cell segmentation methods, and 6) compatibility with formalin-fixed, paraffin-embedded (FFPE) samples. In this study, 980 RNAs and 80 proteins were measured at subcellular resolution in FFPE cultured cell pellets, as well as FFPE tissues from biobanked samples of non-small cell lung cancer (NSCLC) and breast cancer. Cross-platform analysis using 16 cancer cell lines validated high-correlation (R2 ~0.77) and high sensitivity (~1.44 FPKM/TPM; roughly 1 to 2 copies of RNA per cell) when compared to RNA-seq. Real-world archived NSCLC FFPE tumor sections revealed greater than 94% cell detection efficiency for RNA, despite the low RNA quality QV200 20% to the medium quality 65%. The accuracy of protein expression measurements was independent of the level of multiplexing, as demonstrated by the linear behavior of nested multiplexing panels (R2 > 0.9). At 980-plex RNA detection, data analysis allowed identification of over 18 distinct cell types, at least 10 unique tumor microenvironment neighborhoods, and over 100 pairwise ligand-receptor interactions. Data from 8 NSCLC samples comprising over 800,000 single cells and ~260 million transcripts are released into the public domain (www.nanostring.com) to allow for extended data analysis by the entire spatial biology research community.

Highly Efficient Synthetic CRISPR RNA/Cas9-Based Mutagenesis for Rapid Cardiovascular Phenotypic Screening in F0 Zebrafish

The zebrafish is a valuable vertebrate model to study cardiovascular formation and function due to the facile visualization and rapid development of the circulatory system in its externally growing embryos. Despite having distinct advantages, zebrafish have paralogs of many important genes, making reverse genetics approaches inefficient since generating animals bearing multiple gene mutations requires substantial efforts. Here, we present a simple and robust synthetic CRISPR RNA/Cas9-based mutagenesis approach for generating biallelic F0 zebrafish knockouts. Using a dual-guide synthetic CRISPR RNA/Cas9 ribonucleoprotein (dgRNP) system, we compared the efficiency of biallelic gene disruptions following the injections of one, two, and three dgRNPs per gene into the cytoplasm or yolk. We show that simultaneous cytoplasmic injections of three distinct dgRNPs per gene into one-cell stage embryos resulted in the most efficient and consistent biallelic gene disruptions. Importantly, this triple dgRNP approach enables efficient inactivation of cell autonomous and cell non-autonomous gene function, likely due to the low mosaicism of biallelic disruptions. In support of this finding, we provide evidence that the F0 animals generated by this method fully phenocopied the endothelial and peri-vascular defects observed in corresponding stable mutant homozygotes. Moreover, this approach faithfully recapitulated the trunk vessel phenotypes resulting from the genetic interaction between two vegfr2 zebrafish paralogs. Mechanistically, investigation of genome editing and mRNA decay indicates that the combined mutagenic actions of three dgRNPs per gene lead to an increased probability of frameshift mutations, enabling efficient biallelic gene disruptions. Therefore, our approach offers a highly robust genetic platform to quickly assess novel and redundant gene function in F0 zebrafish.

The evolution of splicing: transcriptome complexity and transcript distances implemented in TranD

Alternative splicing contributes to organismal complexity. Comparing transcripts between and within species is an important first step toward understanding questions about how evolution of transcript structure changes between species and contributes to sub-functionalization. These questions are confounded with issues of data quality and availability. The recent explosion of affordable long read sequencing of mRNA has considerably widened the ability to study transcriptional variation in non-model species. In this work, we develop a computational framework that uses nucleotide resolution distance metrics to compare transcript models for structural phenotypes: total transcript length, intron retention, donor/acceptor site variation, alternative exon cassettes, alternative 5'/3' UTRs are each scored qualitatively and quantitatively in terms of number of nucleotides. For a single annotation file, all differences among transcripts within a gene are summarized and transcriptome-level complexity metrics: number of variable nucleotides, unique exons per gene, exons per transcript, and transcripts per gene are calculated. To compare two transcriptomes on the same co-ordinates, a weighted total distance between pairs of transcripts for the same gene is calculated. The weight function proposed has larger penalties for intron retention and exon skipping than alternative donor/acceptor sites. Minimum distances can be used to identify both transcript pairs and transcripts missing structural elements in either of the two annotations. This enables a broad range of functionality from comparing sister species to comparing different methods of building and summarizing transcriptomes. Importantly, the philosophy here is to output metrics, enabling others to explore the nucleotide-level distance metrics. Single transcriptome annotation summaries and pairwise comparisons are implemented in a new tool, TranD, distributed as a PyPi package and in the open-source web-based Galaxy (www.galaxyproject.org) platform.

Circadian Clock Gene Period Contributes to Diapause via GABAeric-Diapause Hormone Pathway in Bombyx mori

Diapause is a developmental transition in insects based on seasonal adaptation to adversity; it is regulated by a circadian clock system and the endocrine system. However, the molecular node and its mechanism underlying the effects of these systems are still unclear. Here, a mutant of Bombyx mori with the circadian clock gene Period (Per) knocked out was constructed, which dramatically changed the classic diapause-destined pathway. Per-knockout silkworms powerfully attenuated, but could not completely block, the predetermined effects of temperature and photoperiod on diapause determination, and this effect depended on the diapause hormone (DH) pathway. The impaired transcription-translation feedback loop of the circadian clock system lacking the Per gene caused direct up-regulation of the expression of GRD, a receptor of γ-aminobutyric acid (GABA), by changing expression level of Cycle. The synthesis of GABA in the tissue complex of brain-suboesophageal ganglion then increased and restricted the decomposition, which continuously promoted the GABAergic signal to play a role, and finally inhibiting (delaying) the release of DH to the hemolymph, and reducing the diapause-inducing effect of DH. The results provided an example to explain the regulatory mechanism of the circadian clock on endocrine hormones in the silkworm.

Adaptation in outbred sexual yeast is repeatable, polygenic, and favors rare haplotypes

We describe the results of a 200 generation Evolve and Resequence (E&R) study initiated from an outbred dipliod recombined synthetic base population derived from 18 genetically diverse founders. Replicate populations were maintained at large effective population sizes (>105 individuals), exposed to several different chemical challenges over 12 weeks of evolution, and whole-genome resequenced. Weekly forced outcrossing implies a per gene per cell-division recombination rate higher than that achieved in Drosophila E&R studies. In 55 sexual populations we observe large fitness gains and highly repeatable patterns of genome-wide haplotype change within each chemical challenge. There was little evidence for pervasive pleiotropy, as evidenced by patterns of haplotype change between drug treatments. Within treatment adaptation appears highly polygenic with almost the entire genome showing significant consistent haplotype change. Finally, adaptation was almost always associated with only one of the 18 founder alleles, suggesting selection primarily acts on rare variants private to a founder or haplotype blocks harboring multiple mutations. This observation contradicts the notion that adaptation is often due to subtle frequency shifts at intermediate frequency variants.

PER Gene Family Polymorphisms in Relation to Cluster Headache and Circadian Rhythm in Sweden

The trigeminal autonomic cephalalgia, cluster headache (CH), is one of the most painful disorders known to man. One of the disorder’s most striking features is the reported diurnal rhythmicity of the attacks. For a majority of patients, the headache attacks occur at approximately the same time every day. Genetic variants of genes involved in the circadian rhythm such as Period Circadian Regulator 1, 2, and 3 (PER1, 2 and 3) are hypothesized to have an effect on the rhythmicity of the attacks. Six PER1, 2 and 3 genetic markers; the indel rs57875989 and five single nucleotide polymorphisms (SNPs), rs2735611, rs2304672, rs934945, rs10462020, and rs228697, were genotyped, using TaqMan® or regular polymerase chain reaction (PCR), in a Swedish CH case control material. Logistic regression showed no association between CH and any of the six genetic variants; rs57875989, p = 0.523; rs2735611, p = 0.416; rs2304672, p = 0.732; rs934945, p = 0.907; rs10462020, p = 0.726; and rs228697, p = 0.717. Furthermore, no difference in allele frequency was found for patients reporting diurnal rhythmicity of attacks, nor were any of the variants linked to diurnal preference. The results of this study indicate no involvement of these PER genetic variants in CH or diurnal phenotype in Sweden.

NGS comparison of Downy and Powdery mildew resistance genomic regions between 'Regent' and 'Red Globe'

Powdery and downy mildew have a large negative impact on grape production worldwide. Quantitative trait loci (QTL) mapping projects have identified several loci for the genetic factors responsible for resistance to these pathogens. Several of these studies have focused on the cultivar Regent, which carries the resistance loci to downy mildew on chromosome 18 (Rpv3), as well powdery mildew on chromosome 15 (Ren3, Ren9). Several other minor resistance loci have also been identified on other chromosomes. Here we report on the re-sequencing of the Regent and Red Globe (susceptible) genomes using next generation sequencing. While the genome of Regent has more SNP variants than Red Globe, the distribution of these variants across the two genomes is not the same, nor is it uniform. The variation per gene shows that some genes have higher SNP density than others and that the number of SNPs for a given gene is not always the same for the two cultivars. In this study, we investigate the effectiveness of studying the variation of non-synonymous to synonymous SNP ratio's between resistant and susceptible cultivars in the target QTL regions as a strategy to narrow down the number of likely candidate genes for Rpv3, Ren3 and Ren9.

Gene Ontology Meta Annotator for Plants (GOMAP)

Annotating gene structures and functions to genome assemblies is necessary to make assembly resources useful for biological inference. Gene Ontology (GO) term assignment is the most used functional annotation system, and new methods for GO assignment have improved the quality of GO-based function predictions. The Gene Ontology Meta Annotator for Plants (GOMAP) is an optimized, high-throughput, and reproducible pipeline for genome-scale GO annotation of plants. We containerized GOMAP to increase portability and reproducibility and also optimized its performance for HPC environments. Here we report on the pipeline’s availability and performance for annotating large, repetitive plant genomes and describe how GOMAP was used to annotate multiple maize genomes as a test case. Assessment shows that GOMAP expands and improves the number of genes annotated and annotations assigned per gene as well as the quality (based on $$F_{max}$$ F max ) of GO assignments in maize. GOMAP has been deployed to annotate other species including wheat, rice, barley, cotton, and soy. Instructions and access to the GOMAP Singularity container are freely available online at https://bioinformapping.com/gomap/. A list of annotated genomes and links to data is maintained at https://dill-picl.org/projects/gomap/.