https://www.um.edu.mt/library/oar/handle/123456789/16097 Thu, 09 Apr 2026 20:28:42 GMT 2026-04-09T20:28:42Z https://www.um.edu.mt/library/oar/handle/123456789/144349 Title: Determining the genotype frequency of the Diego blood group system in Malta Abstract: This study investigated the prevalence of key Diego (Di) blood group antigens (Dia , Dib , Wra , and Wrb ) in Maltese blood donors using a molecular genotyping approach, with the aim of improving rare blood group detection and enhancing transfusion compatibility. A polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP) was developed to genotype four alleles: DI*A (Dia ), DI*B (Dib ), DI02.03* (Wra ), and DI02.04* (Wrb ). High-quality genomic DNA was extracted from donor blood samples, and allele-specific primers were used to amplify target regions of the solute carrier family 4 member 1 SLC4A1 gene. Selected samples were validated through direct DNA sequencing to confirm assay specificity. Additionally, a standardised questionnaire was administered to record donor parental ancestry. The PCR-RFLP method yielded consistent and reproducible results. All individuals tested negative for the rare DI*A and DI02.03* alleles, indicating an absence of the Dia and Wra antigens. The common DI*B and DI02.04* alleles were detected in all samples, suggesting a Di(a-b+) and Wr(a-b+) phenotype across the cohort. Ancestry data showed that the majority of participants reported full Maltese lineage, and allele frequencies were consistent with those expected in European populations. In conclusion, this study provides the first molecular characterisation of the Di Blood Group System in the Maltese population. Although no rare alleles were identified, the developed PCR-RFLP platform proved reliable for Di genotyping and is suitable for integration into routine blood donor screening. These findings establish a reference point for future studies and support the implementation of DNA-based methods to enhance transfusion safety in increasingly diverse populations. Description: M.Sc.(Melit.) Wed, 01 Jan 2025 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/144349 2025-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/143492 Title: Investigating the role of lipopolysaccharide-stimulated monocytes in the development of immune tolerance in community-acquired pneumonia patients Abstract: Introduction: Pneumonia remains a leading cause of morbidity and mortality worldwide. Beyond antibiotic treatment, disease severity hinges on how monocytes balance immune defence and tolerance, a process which may paradoxically worsen outcomes. Background: Emerging evidence links monocyte “tolerance” in CAP to DNA methylation and metabolic shifts, yet why patients differ in cytokine output remains unclear. We hypothesized that transcriptional changes in monocytes underlie this heterogeneity. Methodology: The study included samples from a previously executed prospective observational investigation of CAP patients and control subjects (ELDERBIOME; NCT02928367). Specifically, RNA-seq data of monocytes purified from 75 patients, stimulated with LPS- or not. In addition, TNF-α levels in supernatants were used to stratify samples as LPS responders or non-responders. Read libraries were prepared using the KAPA RNA HyperPrep Kit with RiboErase. Sequencing was performed on the Illumina HiSeq 4000 platform. Bioinformatics included standard quality control metrics, graph-based read alignment, and subsequent DESeq2 modelling, and pathway/network enrichment. Results and Discussion: At padj ≤ 0.01, LPS reshaped expression of 7,033 genes (3,878 upregulated; 3,155 downregulated). Enrichment pinpointed heightened cytokine and interferon pathways, with type I interferons (IFNB1) strongly induced. TNF-α stratification revealed two distinct monocyte states: high responders amplified interferon signalling and HLA class II expression; low responders favoured antioxidant, ECM, and solute transport programmes. Conclusion: These findings uncover transcriptional blueprints explaining patientspecific monocyte behaviour in CAP. Understanding this immune polarity could guide strategies to rebalance hyperinflammation without compromising pathogen clearance. Description: M.Sc.(Melit.) Wed, 01 Jan 2025 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/143492 2025-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/139237 Title: Enhancing the curation of variants of uncertain significance Abstract: The increased demand for genetic sequencing has consequently led to a rise in the number of Variants of Uncertain Significance (VUS), which are classified as such due to insufficient evidence to determine their pathogenicity. These variants lead to incomplete diagnosis for patients, thus potentially causing anxiety and unease due to the uncertainty associated with VUS and may result in unnecessary treatments. The Laboratory of Molecular Pathology and Genetics, Pathology Department, Mater Dei Hospital, situated at the University of Malta, currently utilises software including VarSome and Franklin, to analyse genetic data and provide variant classifications based on the American College of Medical Genetics and Genomics (ACMG) guidelines. Any VUS identified through this analysis are recorded in Excel files which makes management and reclassification challenging. We developed a software, VUSVista, for organised and semi-automated VUS curation by offering ACMG criteria management, referencing of external databases like dbSNP and ClinVar, using standardised Human Phenotype Ontology (HPO) terms to describe sample phenotypes, and linking variants to pseudonymised samples. The absence of personal identifiers in the software safeguards patients’ privacy and makes it GDPR-compliant. VUSVista automatically checks for any updates to ClinVar’s germline classification entries for recorded VUS and searches for newly released publications referencing its VUS using LitVar 2.0. Users receive email notifications whenever a ClinVar entry is updated or a relevant publication is found, which enables them to stay informed about discoveries made by the scientific community. If significant evidence is obtained either from these discoveries or generated internally in the laboratory, users may reclassify the VUS using VUSVista. Our software maintains three audit trails for each of its recorded VUS: one representing any changes applied to the VUS, one for ClinVar update checks and one for publication checks. These audit trails are essential for ensuring traceability and accountability within the diagnostics laboratory’s Quality Management System (QMS). Five individuals working at the aforementioned laboratory participated in one-to-one sessions to evaluate VUSVista and its features. This was followed by a focus group where they discussed the system and shared feedback. During the system’s evaluation, the participants highlighted the automated checks, notification emails, audit trails, and the ability to link variants to samples as significantly useful features that set VUSVista apart from the other systems which they currently use. The participants also suggested improvements to increase the likelihood of adopting the system, such as filtering notifications to only include updates about variants of interest and adding intermediate strengths for ACMG criteria. Overall, VUSVista is a software designed to facilitate VUS curation and reclassification which increases the likelihood of patients receiving a comprehensive diagnosis and in turn, the most accurate management. Description: M.Sc.(Melit.) Wed, 01 Jan 2025 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/139237 2025-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/139236 Title: Identifying founder variants in the Maltese population via identity by descent Abstract: The genetic architecture of populations has been significantly shaped by various evolutionary mechanisms. Bottleneck and founder effects cause an enrichment of variants known as founder variants, and their frequency tends to be higher than expected in certain populations. Founder variants are inherited across generations as part of a haplotype block. This leads to the occurrence of a shared pattern of genetic variation between individuals who share a common ancestor, a process known as Identity-by-Descent (IBD). The small population and geographical position of Malta made the island susceptible to many invasion and migration events, increasing the likelihood of founder effects. One Maltese founder variant has been recorded; related to 5,6,7,8-tetrahydrobiopterin deficiency (QDPR p.Gly23Asp). Identification of more founder variants would allow for a more specific approach in genetic testing of the Maltese population and the development of preventive measures and treatments, saving lives and resources. In this dissertation 1,076 Maltese genomes were used to analyse a list of variants and identify whether IBD segments can be used to determine the variant’s founder status in the Maltese. The benchmarking tool IBD Benchmark was used to test six IBD detection tools and identify the best performing one. This was optimised and used to perform IBD detection on a representative Maltese dataset. A list of variants of interest to the Maltese population was compiled, consisting of 15 variants locally relevant to a variety of disorders. A bioinformatics pipeline was developed to generate horizontal bar plots, heatmaps and variant genetic frameworks for founder variant analysis. Six variants were found to be very likely founder variants of the Maltese population. The heatmaps showed these variants as part of a genetic framework, which is shared among all of the individuals that have the variant. Four variants remained inconclusive upon interpretation of the results, while no IBD segments were detected for five other variants. Having successfully met the key objectives of this study, numerous avenues for further research into founder variants have emerged. The bioinformatics pipeline developed in this project can be applied to any other variant, facilitating the process of identifying founder variants. Description: M.Sc.(Melit.) Wed, 01 Jan 2025 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/139236 2025-01-01T00:00:00Z