Genomics Medicine (Ireland)

Abstract Background Preimplantation genetic testing for monogenic disorders (PGT-M) represents a critical clinical strategy for preventing the transmission of hereditary diseases from carriers to offspring, with its diagnostic efficacy heavily dependent on the accuracy and coverage of detection platforms. Genome-wide SNP array such as the Asian Screening Array (ASA), has demonstrated favorable performance in haplotype analysis for PGT-M. Here, we systematically evaluated the efficiency of the Asian Screening Array-based PGT workflow in PGT application for different genetic disorders. Methods We conducted a retrospective analysis by reviewing 377 pedigrees underwent PGT-M preclinical work-up and 367 PGT-M clinical cycles (1677 embryos in total) based on the Asian Screening Array. We established detection strategies combining ASA haplotyping analysis and individualized direct mutation detection based on different genetic patterns. Long-read sequencing or single-sperm haplotyping strategy was applied for families with de novo pathogenic variants or lacking family member samples. Individualized direct mutation detection methods such as Gap-PCR, RP-PCR, PCR-RFLP were adopted for different cases. Results Results indicated the clinical validity of our ASA-based PGT workflow, integrating linkage analysis, direct mutation detection, and chromosomal CNV screening. The number of SNPs available for linkage analysis in the upstream and downstream regions of target genes/regions is sufficient for most of the cases. Individualized direct mutation detection methods for different cases also validated the ASA haplotyping results. In haplotype analysis, the method based on long-read sequencing is more effective than single-sperm haplotyping strategy. Detailed haplotyping strategy for families with microdeletions and tandem duplications was established based on ASA. A total of 636 embryos were ultimately deemed suitable for transfer after undergoing linkage analysis and CNV detection. Conclusion This study validated the feasibility and superiority of ASA-based approach in PGT, also provided a standardized and reliable technical solution for the clinical prevention of different genetic disorders.

Intratumoral heterogeneity complicates the interpretation of single-test biomarkers. Microsatellite instability (MSI) is one such biomarker, which is used to guide immune checkpoint inhibitor treatment by classifying samples as having high microsatellite instability (MSI-H) or as microsatellite stable (MSS). However, it is unknown whether MSI itself is a heterogeneous phenomenon. To test this, we curated data from several single-cell RNA sequencing studies with clinical MSI status and developed a computational pipeline that quantifies intratumoral heterogeneity in MSI. Out of 49 individuals, 15 showed evidence of divergence in MSI status between clusters of cancer cells, and most had distinct MSI-H and MSS subclones. These results question the use of MSI as a binary biomarker, and we hypothesize that accounting for heterogeneity could improve its use as a predictive biomarker. Further studies are required to determine the frequency of MSI heterogeneity at the population level and whether it can have clinical implications.

Tapered two-mode fiber interferometer with a diameter of 12.5 μm in the cone region is fabricated for label-free DNA detection, with refractive index sensitivity of 1222.59 nm/RIU and ultra-low limits of detection (LOD) of ~ 0.5 aM.

Abstract The genomic representation of populations across the globe is critical to ensuring a comprehensive and equitable human reference. Constructing a pangenome graph reference for different populations is the best approach to addressing local genomic diversities. Although major initiatives across continents are underway to construct pangenome graph references, the field lacks the necessary toolsets for tertiary analysis to characterize telomere-to-telomere (T2T) assemblies and the complexity of haplotypes. PanScan is a bioinformatics software package developed for human pangenome tertiary analysis. It includes multiple modules designed to detect duplicated gene sets from T2T assemblies, identify novel variants and sequences, as well as detect and visualize complex genomic regions through pangenome graph haplotype loops. We have used multiple pangenomes across different populations to assess the tertiary analysis and their accuracy. The tool is designed to streamline tertiary analysis and is compatible with multiple pangenome graph construction algorithms. PanScan is freely available on GitHub (https://github.com/CATG-Github/panscan), where users can provide human pangenome assemblies or VCF files as inputs for automated analyses through command-line operations on Linux systems.Graphical Abstract

Abstract Vimentin is a type III intermediate filament (IF) protein, that is induced in a large number of solid tumours. A single cysteine at position 328 in vimentin plays a crucial role in assembly, organisation and stability of IFs. However, its exact function during epithelial mesenchymal transition (EMT) and cancer progression has not been investigated. To investigate this, we have transduced wildtype (WT) and C328S vimentin separately in MCF-7 cells that lack endogenous vimentin. The expression of C328-VIM impacted vimentin-actin interactions and induced EMT-like features that include enhanced cell proliferation, migration, invasion accompanied by reduced cell adhesion when compared to the wildtype cells. Functional transcriptomic studies confirmed the upregulation of EMT and mesenchymal markers, downregulation of epithelial markers as well as acquisition of signatures associated with cancer stemness (CD56, Oct4, PROCR and CD49f) thus transforming MCF-7 cells from oestrogen positive to triple reduced (ESR1, PGR, and HER2) status. We also observed a stark increase in the expression of long non-coding RNA,XISTin MCF-7 cells expressing C328-VIM. Targeting the mutant vimentin orXISTby RNA interference partially reversed the phenotypes in C328-VIM expressing MCF-7 cells. Furthermore, introduction of C328-VIM cells into nude mice promoted tumour growth by increasing cancer stemness in an oestrogen independent manner. Altogether, our studies provide insight into how cysteine 328 in vimentin dictates mechano-transduction signals to remodel actin cytoskeleton and protect against EMT and cancer growth via modulating lncRNAXIST. Therefore, targeting vimentin and/orXISTvia RNA interference should be a promising therapeutic strategy for breast cancer treatment.Abstract Figure

This abstract was presented as the Food Systems Theme Highlight.The rate of climate change has significantly increased over the last decade, with 2023 reported as the warmest year on record(1). Human activities contribute significantly to greenhouse gas emissions (GHGe) and environmental degradation(1) with food production and consumption contributing up to 37% of global emissions(2). Recognising the power of food service, and food choices, is crucial in addressing these environmental challenges(3). Data on the impact that reformulation has on reducing environmental impact is required to ensure evidence based strategy development and data based decision making. The LIFE Programme Climate Smart Chef (CSC) project aims to engage chefs to promote low-emission, nutritious diets aligned with EU Climate Policy and the Farm to Fork (F2F) Strategy. It targets improved public health, reduced environmental impact, and food waste(4). Under this project, a digital environmental impact tool ‘Foodprint’ was developed by Nutritics. This study aims to analyse the nutritional content and environmental impact of original and reformulated recipes entered by chefs who completed training between July and October 2023.Chefs were required to enter their recipes, undertake training on sustainable menu design and subsequently reformulate their recipes using Foodprint as a post training exercise. 200 recipes were created by the chefs (100 original; 100 reformulated). Nutritics software was used for the analysis, with the LIFE Su-Eatable LIFE database on carbon and water selected for use in the Foodprint tool. The impact of reformulation on nutrition, carbon and water was analysed using SPSS V29.For the environmental analysis, reformulating the recipes had significant reductions in carbon emissions (P = 0.002), (Average −51.18%; Range −6.66 −0.96 kg C02eq), water footprint (P = 0.278), (Average -46.63%; Range −17839L to 4035L). For the nutrition analysis, reductions were also observed for sugar (P = 0.001), (Average −9.42%; Range −16.40g to +24g), salt (P = 0.233), (Average −12.57%; Range −5.42g to +1.80g), and saturated fat (P = 0.043) (Average −39.77%; Range -34.90g to +12.20g).Reformulation resulted in decreased environmental impact, and improved nutrition. This study gives a quantitative indication of how reformulating menus can reduce carbon, while improving the nutrient profile of food offerings. This research contributes valuable insights into the use of software to reformulate menus, the complex impact of recipe reformulation, and its potential to positively impact both health and environmental sustainability. This research indicates the potential of menu reformulation in advancing sustainability goals and promoting healthier food choices. Future analysis should analyse cost, additional environmental impacts, serving size, and demographic analysis to provide more in-depth analysis.

Monocytes, an innate immune cell subtype, influence inflammation and vascular health. Biological sex impacts exercise gene expression in leukocytes collectively, but little is known about specific responses of monocytes in adolescents. We hypothesized, that genomic monocyte responses to exercise would be: 1) related to inflammation and cardiovascular health; and 2) influenced by sex. PURPOSE: To identify monocyte gene expression in response to acute exercise in adolescents. METHODS: 9 females and 12 males (14-17 y/o) performed ten 2-min exercise bouts at constant work of 71% (females) and 72% (males) of maximum work rate, interspersed with 1-min rest. Blood was obtained before and immediately after exercise. Monocytes were isolated (Miltenyi Biotec), and RNA-seq was performed. R package limma (v3.50.3) was used to perform differential gene expression analysis (FDR < 0.1) and DAVID Bioinformatics tool to identify significantly enriched KEGG Pathways (EASE score < 0.05). RESULTS: Exercise altered a greater number of genes in females (5137) compared to males (568). Genes were enriched in COVID-19 gene pathway in both sexes. In the males only: gene pathways were identified in TNF Signaling, Fluid Shear Stress, and Atherosclerosis; in the females: Adipocytokine Signaling, ErbB Signaling, EGFR Tyrosine Kinase Inhibitor Resistance. CONCLUSION: Exercise gene expression in males was 11% of female expression. The COVID-19 pathway was affected in both sexes, as were genes involved in immune regulation, suggesting that exercise modulates inflammation in all adolescents. Pathways linked to vascular health predominated in males, while pathways responsible for adipose cell crosstalk were found in females. Mechanisms responsible for the large sexual dimorphism in gene expression remains unknown. Monocyte gene expression in response to brief exercise may help provide insight and prevention strategies for pediatric origins of adult diseases and lead to sex-specific precision of the exercise prescription in adolescents. NIH Grant No.: U01 TR002004 (REACH project)

Background:Approximately 40% of patients with systemic lupus erythematosus (SLE) develop organ damage within the first 5 years of diagnosis due to uncontrolled disease activity and cumulative steroid exposure, resulting in a greater risk of early mortality.[1] Timely intervention with disease-modifying biologics such as belimumab (BEL) in the early stages of SLE might modify the disease course and prevent irreversible organ damage while avoiding the need for high-dose glucocorticoids (GCs) or immunosuppressants (IS). Previous studies suggested that BEL may have benefit when used earlier in the course of the disease, especially in patients with minimal or no organ damage at baseline who received BEL within 2 years of classification.[2,3] However, the use of BEL early in the treatment pathway has been limited in clinical practice, with the majority of BEL use following the failure of ≥2 IS.Objectives:Present the design of the BeEARLY (BEL in Early SLE) study, which will evaluate the efficacy and safety of BEL in adult SLE patients with early disease.Methods:In this Phase 4, prospective, open-label, single-arm, 3-year clinical study (GSK Study 219240), approximately 350 adult patients with active, autoantibody-positive, early SLE (≤2 years since diagnosis) with no organ damage and with inadequate response to initial standard therapy (ST; with/without conventional IS) will be enrolled. Key eligibility criteria are shown in Table 1. Eligible participants will receive open-label BEL 200 mg/week subcutaneously as an add-on to their initial ST for 52 weeks (Part A), with a long-term extension of up to 104 weeks (end of study is the safety follow-up visit after Week 156; Part B). Key primary and secondary endpoints are shown in Table 2. Participants will be required to taper the average oral GC (OGC; prednisone equivalent) dose from Week 12 to ≤7.5 mg/day by Week 26, and to ≤5 mg/day by Week 48 at the discretion of the investigator if they continue to maintain stable disease control on ≤7.5 mg/day. No changes in OGC dose will be permitted between Weeks 48 and 52. An optional taper of concomitant IS may be attempted after the Week 52 study visit (in participants receiving concomitant IS) if the participant has met Definition Of Remission in SLE (DORIS) criteria for ≥6 months. Safety follow-up will continue for 4 weeks after the clinical study intervention is discontinued. Descriptive summaries will be performed for all endpoints.Results:Data for the primary endpoint are estimated to be available in 2027.Conclusion:This clinical open-label trial will investigate the efficacy and safety of subcutaneous BEL in adults with early, active SLE, using relevant disease activity endpoints such as Lupus Low Disease Activity State (LLDAS)[4], DORIS[5] and Cutaneous Lupus Disease Area and Severity Index (CLASI)[6] achieved with the prednisone equivalent target dose of ≤5 mg/day. The open-label design was selected as several other treatment options are available, and BEL is an approved biological therapy in the population under study in this proposed trial. It is expected that early administration of BEL will benefit participants’ short-term outcomes, such as improvements in disease activity, quality of life, and reduction in OGC use, as well as long-term outcomes, including attainment of remission and prevention of organ damage progression.REFERENCES:[1] Urowitz MB, et al Arthritis Care Res 2012;64:132[2] Gatto M, et al Arthritis Rheumatol 2020;72(8):1314[3] Petri M, et al Ann Rheum Dis 2022;81:323[4] Golder V, et al Lancet Rheumatol 2019;1:e95[5] van Vollenhoven RF, et al Lupus Sci Med 2021;8:e000538[6] Chakka S, et al Ann Transl Med 2021;9(5):431Acknowledgements:The study (GSK Study 21940) was funded by GSK. Medical writing support was provided by Olga Conn, Fishawack Indicia Ltd, UK, part of Avalere Health, and funded by GSK.Disclosure of Interests:Michelle Petri BPR Scientific Advisory Committee, Alexion, Amgen, AnaptysBio, Argenx, AstraZeneca, Aurinia, Biogen, Caribou Biosciences, CVS Health, EMD Serono, Eli Lilly, Emergent Biosolutions, GSK, IQVIA, Janssen, Kira Pharmaceuticals, MedShr, Sanofi, and SinoMab, AstraZeneca, Aurinia, Eli Lilly, Exagen, GSK, Janssen, and Thermofisher, Karen Costenbader GSK, BMS, Amgen, AstraZeneca, Cabaletta Bio, Gilead, Exagen, Merck, Marta Mosca GSK, AstraZeneca, Otsuka, Janssen, AbbVie, Lilly, GSK, AstraZeneca, Otsuka, UCB, AbbVie, Idorsia, GSK, Sophie Collingborn VeramedIs also a complimentary worker on assignment with GSK and a Contractor from Veramed, Roger A. Levy GSK, GSK, Ciara O’Shea GSK, GSK, Holly A. Quasny GSK, GSK.

Abstract Backgroud Dystrophinopathies are the most common X-linked inherited muscle diseases, and the disease-causing gene is DMD. Exonic duplications are common type of pathogenic variants in DMD gene, however, 5’ end exonic duplications containing exon 1 are less common. When assessing the pathogenicity of exonic duplications in the DMD gene, consideration must be given to their impact on the reading frame. Traditional molecular methods, such as multiplex ligation-dependent probe amplification (MLPA) and next generation sequencing (NGS), are commonly used in clinic. However, they cannot discriminate the precise physical locations of breakpoints and structural features of genomic rearrangement. Long-read sequencing (LRS) can effectively overcome this limitation. Results We report three cases involving the duplications of exon 1 and/or exon 2 in DMD gene. The locations of the breakpoints and the characteristics of structural variants (SVs) are identified through LRS, enabling the classification of the variants' pathogenicity. Two distinct variant types encompassing exon 1 in the DMD Dp427m isoform and/or Dp427c isoform are identified, which have been infrequently reported previously. The male individuals harboring duplication variant of consecutive exons 1–2 in the DMD canonical transcript (Dp427m) and exon 1 in the Dp427c transcript are normal, indicating the variant is benign. However, one patient carries a complex SVs involving exon 1 of the DMD Dp427c transcript showing an obvious phenotype. Conclusions Our research sheds light on the complexity of DMD variants encompassing Dp427c/Dp427m promoter regions and emphasizes the importance of cautious interpretation when assessing the pathogenicity of DMD 5' end exonic duplications, particularly in carrier screening scenarios without an affected proband.

&lt;div&gt;Abstract&lt;p&gt;Previously, we reported that a recombinant vaccinia virus (VACV) carrying a light-emitting fusion gene enters, replicates in, and reveals the locations of tumors in mice. A new recombinant VACV, GLV-1h68, as a simultaneous diagnostic and therapeutic agent, was constructed by inserting three expression cassettes (encoding &lt;i&gt;Renilla&lt;/i&gt; luciferase–&lt;i&gt;Aequorea&lt;/i&gt; green fluorescent protein fusion, β-galactosidase, and β-glucuronidase) into the &lt;i&gt;F14.5L, J2R&lt;/i&gt; (encoding thymidine kinase) and &lt;i&gt;A56R&lt;/i&gt; (encoding hemagglutinin) loci of the viral genome, respectively. I.v. injections of GLV-1h68 (1 × 10&lt;sup&gt;7&lt;/sup&gt; plaque-forming unit per mouse) into nude mice with established (∼300–500 mm&lt;sup&gt;3&lt;/sup&gt;) s.c. GI-101A human breast tumors were used to evaluate its toxicity, tumor targeting specificity, and oncolytic efficacy. GLV-1h68 showed an enhanced tumor targeting specificity and much reduced toxicity compared with its parental LIVP strains. The tumors colonized by GLV-1h68 exhibited growth, inhibition, and regression phases followed by tumor eradication within 130 days in 95% of the mice tested. Tumor regression in live animals was monitored in real time based on decreasing light emission, hence demonstrating the concept of a combined oncolytic virus–mediated tumor diagnosis and therapy system. Transcriptional profiling of regressing tumors based on a mouse-specific platform revealed gene expression signatures consistent with immune defense activation, inclusive of IFN-stimulated genes (&lt;i&gt;STAT-1&lt;/i&gt; and &lt;i&gt;IRF-7&lt;/i&gt;), cytokines, chemokines, and innate immune effector function. These findings suggest that immune activation may combine with viral oncolysis to induce tumor eradication in this model, providing a novel perspective for the design of oncolytic viral therapies for human cancers. [Cancer Res 2007;67(20):10038–46]&lt;/p&gt;&lt;/div&gt;