https://www.um.edu.mt/library/oar/handle/123456789/117048 Sun, 07 Jun 2026 17:20:52 GMT 2026-06-07T17:20:52Z https://www.um.edu.mt/library/oar/handle/123456789/120171 Title: Role of canonical WNT pathway genes in osteoporosis & fracture risk Abstract: Osteoporosis is a multifactorial bone disease characterised by reduced bone mass and microarchitectural deterioration, leading to a high fracture risk. It has a strong genetic component, with several gene variants involved in the disease including those within WNT16, DKK1, and SOST, with all three encoded proteins having crucial roles in the WNT/β-catenin signalling pathway. Downstream to SOST is a distal enhancer element known as ECR5 required for SOST transcriptional activity. The study aimed to determine whether gene variants within WNT16, DKK1, and SOST, including SOST ECR5, alter osteoporosis and fracture risk in Malta. A total of eight carefully selected, BMD-associated variants were genotyped by quantitative real-time PCR (i.e., TaqMan® fluorogenic 5’ nuclease allelic discrimination assay or kompetitive allele specific PCR [KASPTM]) in 1,045 postmenopausal women from the MOFS collection to replicate findings from literature. The SOST ECR5 region was also sequenced using Sanger sequencing in a subset of the individuals with extreme bone phenotypes (i.e., normal to high bone mass, and low bone mass; n = 212) from the MOFS collection, which identified two variants that were subsequently tested in the entire collection. Genotype-phenotype associations were assessed using the Kruskal-Wallis and Mann-Whitney U tests, whereas exposure odds ratios were computed using logistic regression adjusted for confounders. Genotype and allele frequencies of all genotyped variants were in line with European non-Finnish population from gnomAD. Results showed that the homozygous alternative genotype for WNT16 rs142005327 exerted a protective effect on LS BMD (adj-OR: 0.3 [95% CI 0.2-0.7], p=0.003) FN BMD (adj-OR: 0.5 [95% CI 0.3-0.8], p=0.009), all type of low trauma fractures (adj-OR: 0.5 [95% CI 0.3-0.9], p=0.023), and wrist fractures (adj-OR: 0.4 [95% CI 0.1-0.9], p=0.036), together with higher sALP levels (adj-OR: 1.9 [95% CI 1.1-3.3], p=0.027). SOST rs4792909 exhibited a deleterious effect on FN BMD (sBMD GT p=0.012; T-score GT p=0.010), and TH BMD (sBMD GT p=0.021; T-score GT p=0.019), while SOST rs8064375 exhibited a deleterious effect on LS BMD (T-score GA p=0.045). To date, this is the first study linking SOST rs8064375 with BMD. Haplotypes for DKK1 variants were found associated with FN BMD, while WNT16 and SOST haplotypes were associated with LS and FN BMD, and fracture risk. The results showcase the potential role of these variants as genetic determinants of BMD and fractures in Malta, further highlighting the importance of the WNT/β-catenin pathway in bone and pathophysiology. Description: M.Sc.(Melit.) Sun, 01 Jan 2023 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/120171 2023-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/119532 Title: The genetics of hypermobile Ehlers-Danlos syndrome : a local study Abstract: Background: The Ehlers-Danlos Syndromes (EDS) are a collection of thirteen rare hereditary connective tissue disorders which present with heterogeneous phenotypes. The commonest and less severe subtype, hypermobile Ehlers-Danlos Syndrome (hEDS), presents a vast spectrum of signs and symptoms, including extensive joint hypermobility, musculoskeletal instability, chronic pain and autonomic dysfunction. To date, various international research efforts failed to uncover its molecular genetic aetiology, and this is further confounded by inter-familial genetic heterogeneity. Aims: This original local research study sought to elucidate the genetic aetiology of hEDS in a small group of hEDS patients in Malta and to determine the effect of hEDS on bone mass. Methodology: A multigenerational Maltese family as well as an unrelated participant were recruited. High-throughput sequencing was performed on selected participants using Illumina technology. Genetic analysis was also supplemented with bone mineral density (BMD) scans and testing of serum bone-related analytes. Results: Two research participants had low bone mass and were classified as osteopenic, while serum levels of bone-related analytes were within the physiological range except in one osteopenic participant. Sequencing data was filtered using an in-house filtering pipeline to obtain a list of genetic variants found segregating with the disease phenotype in the family. This yielded five single nucleotide variants, namely TNXB rs61746206 (c.745G>A, p.Glu249Lys), TNXB rs140304758 (c.6973G>A, p.Val2325Ile), SMAD3 rs189286879 (c.206+32287G>A), FANCA/ZNF276 rs201316239 (n.47G>A) and PNPLA1 rs45524833 (c.745G>A, p.Glu249Lys), which were not detected in the un-related participant. These variants were further annotated using gene databases and in-silico prediction tools to determine their clinical relevance. Discussion: None of these variants was found to be pathogenic, thus highlighting the need for further research on larger cohorts and families of hEDS patients. The discovery of a potentially causative genetic pathogenic variation will enable the establishment of an early diagnosis and appropriate clinical management of this multisystemic hereditary disorder. Description: M.Sc. Biomed. Sc.(Melit.) Sun, 01 Jan 2023 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/119532 2023-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/119531 Title: Unravelling the globin gene switch mechanism in patients with hereditary persistence of foetal haemoglobin Abstract: Haemoglobinopathies are widely recognised as one of the most common monogenic diseases globally, representing a significant global health issue. Hereditary persistence of foetal hemoglobin (HPFH) is a benign genetic condition characterised by continuous production of high levels of foetal hemoglobin (HbF) throughout adulthood, resulting from disrupted globin gene switching. Clinical investigations and molecular findings have demonstrated that the presence of HPFH in conjunction with other haemoglobinopathies reduces the severity of associated symptoms, attributed to elevated levels of HbF. This study focused on three Maltese families, encompassing 11 individuals affected by HPFH due to a truncation mutation (p.K288X) in the KLF1 gene and 11 healthy relatives serving as controls. The primary objective was to gain insights into the underlying genetic and molecular mechanisms involved in the regulation of globin gene switching. Whole genome sequencing (WGS) was performed using DNA extracted from 11 affected individuals and 9 healthy controls. A total of 205 unique variants following a dominant inheritance pattern were identified and found to be present in all affected individuals. All these variants were discovered to be located on chromosome 19 in close proximity to the KLF1 gene. Furthermore, novel variants were discovered in the LMO2 and KLF1 genes, which potentially contribute to the onset of HPFH. A subset analysis focusing on four subjects from Fam F1, exhibiting the highest HbF levels (>3%), revealed variants in the NLRP3 gene and in the RPS9 gene when following autosomal dominant and recessive inheritance patterns, respectively. These variants likely account for the sustained elevation of HbF levels in Fam F1, despite carrying the same KLF1 mutation as other families. Flow cytometry data analysis confirmed the role of KLF1 in the regulation of antigen expression, as individuals with HPFH exhibited reduced levels of BCAM, CD44, and P1 antigens on erythrocytes. Furthermore, analysis of globin gene mRNA expression, revealed that healthy controls had elevated mRNA levels of adult α-, β-globin genes, while individuals with HPFH exhibited higher mRNA levels of the fetal Aγ-globin gene. Proteomic analysis using mass spectrometry (MS) supported these findings and additionally identified 53 proteins significantly correlated with HbF levels in HPFH-affected subjects, suggesting their involvement in globin gene regulation. In conclusion, this study highlights the importance of adapting an integrative approach to understand the molecular mechanisms underlying KLF1 deficiency. The identification of potentially causal variants associated with HPFH, provides valuable insights into the onset of this condition. Further investigations involving functional work to confirm the precise impact of these variants, as well as the role of the identified proteins in the upregulation of HbF levels, can provide a more comprehensive understanding of the underlying genetic architecture of HPFH. Description: M.Sc. Biomed. Sc.(Melit.) Sun, 01 Jan 2023 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/119531 2023-01-01T00:00:00Z https://www.um.edu.mt/library/oar/handle/123456789/119530 Title: Identifying a genetic association between non-autoimmune diabetes & bone mass Abstract: Introduction & Aim: Diabetes and osteoporosis are two common complex diseases that have a strong genetic and environmental component. The genetics of these diseases have been widely studied separately but not as extensively together. This project aims to elucidate the genetics underlying non-autoimmune diabetes and bone mineral density (BMD) through two study approaches; whole exome sequencing (WES) in cases with suspected monogenic diabetes participants and genotyping of two large collections of five literature derived variants. Methods: Five variants derived from the literature associated with both diabetes and BMD were selected for replication in two Maltese case-control studies (MOFS, n = 1174 and T2D, n = 773 collections). Ten non-obese probands with atypical non-autoimmune diabetes diagnosed before the age of 40 years were recruited. Lifestyle, biochemical data and BMD measurements were obtained and WES was carried out to identify possibly deleterious variants. Results: Significant associations between the five genotyped variants and bone and T2D phenotypes were observed, the most notable being within TNFRSF11B rs4876868, whereby the alternative allele was both deleterious for BMD at the TH and LS, and protective for T2D. The alternative allele of variant ADCY5 rs56371916 also appeared to increase fracture risk whilst increasing BMD at the LS and TH, indicating a possible alteration of bone microarchitecture. A polygenic risk score indicated that the five genotyped variants had a negative correlation with HOMA-IR in individuals without T2D and a positive correlation with BMI and HDL-c in T2D individuals. WES of ten probands identified a total of sixteen SNVs and InDels with a possible association with BMD, T2D or both. A missense variant in the GCK gene (c.1364T>A), was identified in one participant with accompanying osteoporosis at the LS, hinting at a potential detrimental effect on bone mass. Two participants also carried a rare frameshift variant in the WRAP53 gene (c.18_19delTC), indicating a likely association with T2D. Conclusion: A genetic link between BMD and T2D was established in literature derived variants and sixteen potentially pathogenic variants are identified through WES. Further confirmatory and functional studies for the shortlisted variants identified through WES are necessary in order to fully elucidate their effect on these two complex diseases. Description: M.Sc. Biomed. Sc.(Melit.) Sun, 01 Jan 2023 00:00:00 GMT https://www.um.edu.mt/library/oar/handle/123456789/119530 2023-01-01T00:00:00Z