OAR@UM Community:

Validation of autosomal dominant polycystic kidney disease variants using long-range pcr and third generation sequencing

Thu, 01 Jan 2026 00:00:00 GMT

Title: Validation of autosomal dominant polycystic kidney disease variants using long-range pcr and third generation sequencing Abstract: Autosomal dominant polycystic kidney disease (ADPKD) is a hereditary disorder which is characterized by the formation and progressive enlargement of multiple bilateral kidney cysts with an estimated prevalence of 1 in 1000 to 1 in 2500 individuals. ADPKD is also associated with a high phenotypic variability even between affected members of the same family such that the age of onset and the severity of the disease differ significantly between affected individuals. With molecular testing, it is known that 80-85 % of the affected individuals possess a pathogenic variant in PKD1 and almost all the rest are characterized by a pathogenic variant in PKD2. This study involved 3 objectives. The first objective was to confirm the presence, location and pathogenicity of the PKD1 p.C508R variant. Long range (LR) PCR followed by long-read 3rd generation amplicon sequencing were performed which successfully determined that the p.C508R variant is present on PKD1P3 in both ADPKD patients and in non-PKD individuals and thus should be reclassified as a likely benign polymorphism. The second objective was to confirm previously identified variants which were shortlisted by 2nd generation high-throughput sequencing (HTS) in the Malta NGS Project. LR-PCR and/or nested PCR followed by Sanger sequencing were used, which successfully confirmed 7 out of 11 potential causative variants. These variants were re-evaluated to determine pathogenicity. The third and final objective of this project was to carry out further testing on the patients from the 2 pedigrees (P12 and P13) in which no potential causative variant had been identified. PacBio whole genome sequencing (WGS) was performed on a single Sequel IIe SMRT flowcell however, unsatisfactory results were obtained, with insufficient coverage across several parts of the genome and almost no coverage across PKD1. Additional sequencing runs on other Sequel IIe SMRT flowcells per genome or higher throughput HiFi systems such as Revio or Vega are necessary. This study highlights the differences between short-read and long-read sequencing techniques and encourages the integration of LR-PCR with long-read 3rd generation sequencing especially for complex genomic regions or genes with homologous pseudogenes such as PKD1. Description: M.Sc.(Melit.)

Deciphering the compartmentalized host response in sepsis secondary to community-acquired pneumonia

Thu, 01 Jan 2026 00:00:00 GMT

Title: Deciphering the compartmentalized host response in sepsis secondary to community-acquired pneumonia Abstract: Sepsis, currently defined as a complex condition resulting from a dysregulated host response to infection leading to lethal organ dysfunction, is a major global health concern and a leading cause of both morbidity and mortality worldwide. Emerging evidence suggests that immune compartmentalization, variation in the immune response between the circulation and the tissue microenvironment, plays a vital role in the clinical trajectory of sepsis. In sepsis secondary to community-acquired pneumonia (CAP), distinct immune cell distribution and localization of cytokine production may contribute to divergent inflammatory signatures between bronchoalveolar lavage fluid (BALF) and plasma. This project aims to identify compartment-specific immune responses which would aid in further uncovering the pathophysiology of pneumosepsis. This project was a prospective observational single-center study in the intensive care unit (ICU) within Mater Dei Hospital, Malta, forming part of the Molecular Endotype-Specific Dynamics of Lung Endothelial Barrier Integrity in Sepsis (MENDSEP) study. Peripheral blood and BALF samples were obtained from 29 consenting critically ill patients diagnosed with sepsis secondary to CAP (pneumosepsis) between 2023 and 2025. In addition, 16 age, sex, and co-morbidity-matched controls were recruited from the community and from St Vincent de Paul Residence (SVPR). The levels of various cytokines and markers in blood and BALF of septic samples were quantified using multiplex immunoassays. Cytokine analysis revealed marked immune compartmentalization in pneumosepsis. Pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), anti-inflammatory cytokines (IL-10) and anti-viral interferons (IFN-α, IFN-β, IFN-γ) were all found to be significantly enriched in BALF relative to plasma (p<0.001), indicating localized inflammatory and antiviral activity. Conversely, angiopoietin-2 (Ang-2) and the Ang-2:Ang-1 ratio were elevated in the circulation, representing systemic endothelial dysfunction. These findings underscore the compartmentalized nature of the immune response in sepsis, with localized pulmonary inflammation alongside systemic endothelial dysfunction. This study lays the groundwork to further elucidate the complex pathophysiology of sepsis, in improving early diagnosis, and the development of compartment-specific therapeutic targets. Description: M.Sc.(Melit.)

Bone fragility and osteoporosis in children and young adults

Mon, 01 Jan 2024 00:00:00 GMT

Title: Bone fragility and osteoporosis in children and young adults Authors: Formosa, Melissa Marie; Christou, Maria A.; Mäkitie, Outi Abstract: Osteoporosis is a metabolic bone disorder which increases fragility fracture risk. Elderly individuals, especially postmenopausal women, are particularly susceptible to osteoporosis. Although rare, osteoporosis in children and young adults is becoming increasingly evident, highlighting the need for timely diagnosis, management and follow-up. Early-onset osteoporosis is defined as the presence of a low BMD (Z-score of ≤ −2.0 in individuals aged < 20 years; T-score of ≤ −2.5 in those aged between 20 to 50 years) accompanied by a clinically significant fracture history, or the presence of low-energy vertebral compression fractures even in the absence of osteoporosis. Affected children and young adults should undergo a thorough diagnostic workup, including collection of clinical history, radiography, biochemical investigation and possibly bone biopsy. Once secondary factors and comorbidities are excluded, genetic testing should be considered to determine the possibility of an underlying monogenic cause. Defects in genes related to type I collagen biosynthesis are the commonest contributors of primary osteoporosis, followed by loss-of-function variants in genes encoding key regulatory proteins of canonical WNT signalling (specifically LRP5 and WNT1), the actin-binding plastin-3 protein (encoded by PLS3) resulting in X-linked osteoporosis, and the more recent sphingomyelin synthase 2 (encoded by SGMS2) which is critical for signal transduction affecting sphingomyelin metabolism. Despite these discoveries, genetic causes and underlying mechanisms in early-onset osteoporosis remain largely unknown, and if no causal gene is identified, early-onset osteoporosis is deemed idiopathic. This calls for further research to unravel the molecular mechanisms driving early-onset osteoporosis that consequently will aid in patient management and individualised targeted therapy.

Idiopathic hypogonadotropic hypogonadism in the Maltese island population : a spectrum of phenotypes and genotypes

Fri, 01 Jan 2021 00:00:00 GMT

Title: Idiopathic hypogonadotropic hypogonadism in the Maltese island population : a spectrum of phenotypes and genotypes Authors: Axiak, C.; Pleven, Adrian; Borg Carbott, Francesca; Attard, Ritienne; Cassar, Karen; Gruppetta, Mark; Vassallo, Josanne; Bezzina Wettinger, Stephanie; Farrugia, Rosienne Abstract: Idiopathic Hypogonadotropic Hypogonadism (IHH) is a rare, genetically heterogeneous infertility disorder characterized by absent or incomplete sexual maturation by age 18. This is due to a deficiency or absence of either pulsatile hypothalamic secretion of gonadotropin-releasing hormone (GnRH) and/or gonadotropins from the anterior pituitary in the presence of low serum sex steroids. IHH diagnosis is only conclusive if there are no other abnormalities to hypothalamic and pituitary morphology and function.