OAR@UM Collection:

Identifying genetic determinants of osteoporosis in Malta

Sat, 01 Jan 2022 00:00:00 GMT

Title: Identifying genetic determinants of osteoporosis in Malta Abstract: Introduction & Aim: Osteoporosis is a bone disease with a strong genetic background. The aim of the study was to identify the underlying genetic cause of osteoporosis in a Maltese pedigree. Methodology: A 3-generation family of 9 relatives aged 21-76 years was recruited. Osteoporosis was defined by spine and hip T-scores or Z-scores derived from BMD measurements by DXA. The proband, a 52-year-old female, was diagnosed with osteoporosis at the hip (T-score FN -3.4; TH -2.7). Whole genome sequencing was performed on 6 relatives followed by sequential variant filtering. The shortlisted variants were tested using Competitive Allelic Specific PCR in the Malta Osteoporotic Fracture Study (MOFS) consisting of a case-control collection of 1,045 Maltese postmenopausal women and other independent families. Results: Two zygosity filtering scenarios following a dominant inheritance pattern identified 6 conserved missense variants, including STAT4 c.1309C>T, CXCR1 c.938G>A, TMEM151B c.545A>G, SLC5A10 c.287A>T, CYP1B1 c.182G>A and SUCLG1 c.236G>A, and 2 nonsense variants PCDHA7 c.2257C>T and FAM227B c.621G>A. The novel variants STAT4 c.1309C>T and TMEM151B c.545A>G were monomorphic in the MOFS. Heterozygosity for the PCDHA7 c.2257C>T was linked to a 3-fold increased risk of osteoporosis at the FN (Adj OR: 3.0 [95% CI 0.9-9.5], p=0.080) in line with the phenotype observed in affected relatives. However, the association did not reach statistical significance possibly due to the small sample size. The alternative allele frequency of the SLC5A10 c.287A>T (T=4%) and SUCLG1 c.236G>A (A=3%) variants was much higher compared to the European non-Finnish population in gnomAD suggesting the possibility of a founder effect. These two variants were detected in affected relatives from another independent 3-generation Maltese family, with heterozygosity for the SUCLG1 c.236G>A variant associated with a protective effect in the case-control collection (Adj OR: 0.5 [95% CI 0.2-10], p=0.034), possibly as a result of a small sample size and genotyping bias. Trends were observed with low BMD for the remaining variants (CXCR1 c.938G>A, CYP1B1 c.182G>A, FAM227B c.621G>A), yet none reached statistical significance especially in view of the low alternative allele frequency (<1%). Conclusion: The combination of the 4 variants from each scenario may possibly be the underlying causal determinants of familial osteoporosis in this 3-generation family. Some trends observed are suggestive of the variants’ role in the genetic architecture of bone disease, possibly via signalling pathways known to be involved in bone homeostasis, such as the WNT/β-catenin pathway and JAK-STAT pathway. Description: M.Sc.(Melit.)

Identification of genetic determinants underlying familial osteoporosis

Sat, 01 Jan 2022 00:00:00 GMT

Title: Identification of genetic determinants underlying familial osteoporosis Abstract: Introduction & Aim: Osteoporosis is a skeletal disease with a strong genetic influence. The study aimed to identify gene variants underlying early-onset osteoporosis in a nuclear Maltese pedigree. Methods: A two-generation family consisting of a 60-year-old affected father, a non-affected mother (54 years) and 2 affected male siblings (aged 29 and 32 years) was recruited through an interviewer-led questionnaire. Osteoporosis was defined by DXA measurements of the spine and hip, and secondary causes of osteoporosis were excluded through biochemical testing. The proband was a 29-year-old male with early-onset osteoporosis having sustained bilateral femur fractures before the age of 19 years. WGS was performed on the Illumina NovaSeq 6000 platform with different modes of inheritance investigated by sequential variant filtering and curation using human tissue and animal expression databases, population studies, databases of protein interactions and signalling cascades as well as in silico protein modelling. The shortlisted variants were genotyped in a population-based case-control collection of 1,045 Maltese postmenopausal women and three other independent affected Maltese families from the Malta Osteoporotic Fracture Study (MOFS) using real-time PCR. Genotyping data from the population study was assessed for genotype and allele frequencies, genotype-phenotype associations using the Mann-Whitney U test and exposure odds ratios by logistic regression adjusted for confounders. Results: A total of 9 conserved missense variants were identified following a dominant inheritance pattern, 2 of which were also predicted to alter pre-mRNA splicing, including METTL11B c.38G>T, NBEAL2 c.1948G>A, ADAM8 c.376G>A, TENM4 c.5934C>G, ACACB c.526G>A, PAPLN c.589G>T, ATP8B1 c.3017A>T, ABCA7 c.2326G>A, and PLIN3 c.319C>A. Two other variants, SUSD5 c.745C>A and SUSD5 c.172C>T were also detected following a compound heterozygous inheritance pattern. The alternative allele frequency of most variants was of <1% in line with gnomAD. Heterozygosity for TENM4 c.5934C>G was associated with a lower lumbar spine (LS) T-score (p=0.036) and LS BMD (p=0.034). Women with the GA genotype for the ABCA7 c.2326G>A had lower levels of serum calcium (p=0.045). Heterozygosity for SUSD5 c.745C>A was associated with lower total hip (TH) T-score (p=0.044), TH BMD (p=0.037) and femoral neck (FN) BMD (p=0.029). PLIN3 c.319C>A was associated with a protective effect on LS BMD (Adjusted OR: 0.36 [95% CI 0.15-0.85], p=0.02) which could be a result of the small sample size and genotyping bias. None of the variants were detected in the other Maltese families. Conclusion: The identified gene variants, alone or in combination, might play a role in the pathogenesis of early-onset osteoporosis with varying effect sizes. The encoded proteins are involved in pertinent bone signalling cascades, including Wnt/β-catenin, RANK-RANKL and Notch signalling, amongst others. Further genotyping in larger collections and extended families is warranted to confirm causality, whereas functional studies of the novel PAPLN c.589G>T and ATP8B1 c.3017A>T splicing variants is required. Description: M.Sc.(Melit.)

Genetics of autosomal dominant polycystic kidney disease

Sat, 01 Jan 2022 00:00:00 GMT

Title: Genetics of autosomal dominant polycystic kidney disease 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. It is a relatively uncommon disorder 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 differs significantly between affected individuals. Through the use of molecular testing, it is known that 80-85 % of the affected individuals possess a pathogenic variant in PKD1 and 15-20 % of the cases are characterized by a pathogenic variant in PKD2. Locally, the Malta Next Generation Sequencing Project is the first study that is being carried out to identify pathogenic variants responsible for causing ADPKD in the Maltese population. High throughput sequencing (HTS) has been performed on 18 unique pedigrees which include 15 affected adults and 3 nuclear families. Candidate variants have been identified in 16 pedigrees. However, HTS data on its own is not sufficient to determine whether the identified variants are pathogenic or benign. Therefore, this study is aiming to carry out a population study where approximately 600 cord blood DNA samples are tested to determine the allele and genotype frequencies of the selected variants using real time genotyping. The Malta NGS Project (n=179) has 105 unrelated HTS datasets and an additional 74 HTS datasets of relatives of probands. The PKD1 p.C508R polymorphism was identified in 13 individuals from the NGS study, 11 of whom were adults from 4 unrelated families without ADPKD and the remaining 2 were ADPKD patients (pedigrees 2 and 10; Cini Masini 2021). In both pedigrees, another potentially pathogenic PKD1 variant apart from the SNP of interest was found. The PKD1 p.L337Q polymorphism was identified in 2 individuals who were both ADPKD patients (pedigrees 5 and 11; Cini Masini 2021). In both pedigrees, other potentially pathogenic PKD1 variants apart from the SNP of interest were also found and, in pedigree 5, the PKD1 p.L337Q was only found in 1 of 3 family members with ADPKD. Based on the findings of this project, it can be concluded that both PKD1 p.L337Q and PKD1 p.C508R are not pathogenic, hence do not contribute to cystogenesis in ADPKD. Description: B.Sc. (Hons)(Melit.)

Colonization of hospital sinks with multi-drug resistant Gram-negative bacteria

Sat, 01 Jan 2022 00:00:00 GMT

Title: Colonization of hospital sinks with multi-drug resistant Gram-negative bacteria Abstract: Water is an essential component of life and it is used on a daily basis for multiple purposes. However, water can easily get contaminated with bacteria. Such contamination is particularly worrying in water systems within healthcare facilities. Apart from the fact that these bacteria can easily end up infecting hospitalized patients, these bacteria can also become antibiotic resistant. This is a phenomenon whereby bacteria can acquire resistance towards certain antibiotics and thus become more difficult to treat. The stronger the use and administration of antibiotics to patients, the more likely, bacteria are to acquire resistance by means of mutations that arise in the bacterial Deoxyribose Nucleic Acid (DNA). Although waterborne bacteria can potentially colonize different surfaces, the main focus of this thesis deals with the colonization of hospital sinks. During this study, a total of 78 sinks in 11 wards within Mater Dei Hospital (MDH) were sampled. Extended spectrum beta lactamases (ESBL) and Carbapenem Resistant Enterobacterales (CRE) producing Gram negative organisms, showing different types of resistance patterns, were isolated (46% of the sinks sampled). Furthermore, the relationship between the organisms that caused infections within the patients and the organisms that were isolated from the sinks of the same ward containing the infected patients, was assessed. Several mitigation strategies are proposed. Some of these strategies are already being used locally by the infection control unit whereas others may be implemented in the near future. Description: B.Sc. (Hons)(Melit.)