Research conducted by some members of the Department of Applied Biomedical Science, headed by Professor Angela Xuereb, has contributed to novel gene discovery in the field of genetics of osteoporosis in postmenopausal women caused by low bone mineral density. A region on chromosome 11p12 was linked to a highly penetrant form of osteoporosis in an extended Maltese family (Vidal et al, 2007). Further work identified CD44 variants that influence gene splicing (Vidal et al, 2009). This identification of genes contributing to the debilitating disease is crucial in identifying susceptible individuals, thus preventative measures can be taken at a younger age, for example, increased physical activity and increased calcium and vitamin D intake. Also, this research aids in the development of new treatments and medications.

A collection of samples and data is currently being set up for the study of osteoporotic fractures, a common cause of morbidity in older adults. This will enable research to be carried out on the genetic causes of fractures that are independent of bone mineral density, thus improving current prediction models of fracture risk based on bone mineral density. Efforts to identify genetic markers associated with osteoporotic fractures are warranted to minimise this devastating health problem, especially prevalent in the elderly community.

More effective treatment and prevention strategies can be developed and improved testing methods can be introduced. This research can also be used to educate the community so that 'in the future as many people as possible have stronger bones in later life'.

References
Vidal, C., Cachia, A. & Xuereb-Anastasi, A. (2009) Effects of a synonymous variant in exon 9 of the CD44 gene on pre-mRNA splicing in a family with osteoporosis. Bone, 45, 736-742.
Vidal, C., Galea, R., Brincat, M. & Xuereb Anastasi, A.(2007) Linkage to a region on chromosome 11p12 in two Maltese families with severe osteoporosis. European Journal of Human Genetics 15, 800 - 809.

Study coordinator: Dr Stephanie Bezzina Wettinger

The Maltese Acute Myocardial Infarction (MAMI) Study was started in 2010 and is still ongoing. The MAMI Study Collection includes 423 cases with a first MI (before age 70 in males and 75 in females), 465 population controls (frequency matched by gender and age groups) and 210 relatives of cases (mainly siblings, and some adult children). The inclusion of relatives in a case-control study is very rare and increases the potential of genetic studies. An important aspect of genetic and epidemiological studies is the quality of the collected data and samples, thus blood samples were processed, aliquoted and stored within an hour of collection. Particular attention was paid to pre-analytical variables ensuring that the sample collection is imminently suitable for studies aimed at identifying biomarkers; particularly circulating non-coding RNA biomarkers. Furthermore, DNA, RNA, serum and plasma (EDTA and citrate) has been banked for future studies. This is a state-of-the-art collection with stringent quality control procedures and sample processing times to ensure the highest quality of the samples. Over 130 variables were measured in all samples including biochemical, haematological, coagulation, immunological and physical parameters besides ECG results and RNA profiling of 55 transcripts relating to inflammation. Extensive data was also collected from an interviewer-led questionnaire that covered details on lifestyle and environmental factors, (including smoking, alcohol use, occupation, stress, sleeping pattern, diet, socioeconomic status), use of medications, family history and medical history as well as a thorough review of medical records (including retrieval of past laboratory test results). Much of this data has been analysed, one variable at a time, in a conventional manner. The known risk factors of MI were similar to those observed in other studies. HTS has been carried out on selected extreme phenotypes based on findings from the laboratory tests conducted. This collection and its characterisation were funded by a national R&I grant (2008 programme) and national scholarship grants. It is a collaborative effort between the Department of Applied Biomedical Science, UM, and the Maltese Department of Health, besides collaborations with the Academic Medical Centre in Amsterdam and the University of Twente. Ethics approval for this collection includes HTS testing and open consent; and selected gene panel sequencing and RNASeq are currently being carried out. Funding has now been secured to conduct whole genome sequencing and transcriptomics on the entire collection as combining HTS data with the extensive phenotype, clinical and biochemical data available will be a priceless resource enabling innovative bioinformatics approaches to be developed and applied, based on pathway analysis, systems biology and reverse phenotyping, focussing on gene-gene and gene-environmental interactions.

References

Attard R, Farrugia R, Bezzina Wettinger S. The preanalytical phase starts before blood collection; Biochemica Medica 2013;23(1): A27

Attard Ritienne, Dingli Philip, Cassar Karen, Doggen Carine, Farrugia Rosienne, Bezzina Wettinger Stephanie. Behavioural aspects of smoking (both passive and active) and alcohol consumption on the risk of myocardial infarction; Atherosclerosis, 2014: Vol 235, Issue 2, page e213

Attard Ritienne, Vassallo Josanne, Dingli Philip, Cassar Karen, Doggen Carine, Farrugia Rosienne, Bezzina Wettinger Stephanie. Controlled versus uncontrolled diabetes and the risk of myocardial infarction;  Atherosclerosis, Vol 235, Issue 2, Pages e69-e70

Attard R, Dingli P, Doggen C, Cassar K, Farrugia R, Bezzina Wettinger S. Socioeconomic and Psychosocial Risk Factors of Myocardial Infarction; Atherosclerosis; 2015: 241, e58-e59

Dingli P, Attard R, Cassar K, Doggen C, Farrugia R, Bezzina Wettinger S. Persistenly Elevated Risk of Myocardial Infarction After Smoking Cessation. Do Lipids Play a Role?; Dec 2014

Attard R, Dingli P, Doggen CJM, Cassar K, Farrugia R, Bezzina Wettinger S. The impact of frequency, pattern, intensity, and type of alcohol consumption, and its combined effect with smoking on inflammation, lipid profile, and the risk of myocardial infarction; J Public Health (Berl.) 2019, doi:10.1007/s10389-019-01172-3.

 Attard R, Dingli P, Doggen CJM, Cassar K, Farrugia R, Wettinger SB, The impact of passive and active smoking on inflammation, lipid profile and the risk of myocardial infarction;  Open Heart 2017, Aug 8;4(2): e000620. DOI: 10.1136/openhrt-2017-000620.

Pharmacologically mediated regulation of the expression of the human gamma (γ) globin genes could be a potential therapeutic approach for haematological disorders particularly beta (β) thalassaemia and sickle cell disease (SCD). It is a well-documented fact that an increase of foetal haemoglobin (Hb F) leads to a considerable improvement of the clinical status of the patients affected by these haemoglobinopathies. Reactivating the γ-globin gene would greatly ameliorate the symptoms of β-haemoglobinopathies. Various studies have shown that three important genes; the v-myb myeloblastosis viral oncogene homolog (MYB), Bcell lymphoma/leukemia 11A (BCL11A) gene and Erythroid Krüppel-Like Factor  (KLF1) gene have an impact on the regulation of Hb F. In this study erythroid progenitor cells are cultured from patients with KLF1 mutations, β-thalassaemia and normal healthy adults as controls. The cells are subjected to 3 types of compounds, hydroxyurea, thalidomide and 5’azacytidine and determine which combination of drug to patient cells works optimally to augment Hb F.

References
Borg, J., et al (2011) Erythroid phenotypes associated with KLF1 mutations. Haematologica 96(5):635-638.
Borg, J., et al., (2010) Haploinsufficiency for the Erythroid Transcription Factor KLF1 Causes Hereditary Persistence of Foetal Haemoglobin. Nature Genetics. 42, 801-805.

Hereditary Persistence of Foetal Haemoglobin (HPFH) is when individuals  exhibit a higher than normal (> 2%) foetal haemoglobin (Hb F). HPFH has been widely investigated for its role as a model to understand better Hb F regulation and control. Thus far three important genes appear to regulate this control and include the MYB proto-oncogene, KLF1 and BCL11A genes. Individuals with certain types of haematological malignancies such as Congenital Dyserythropoietic Anaemia (CDA), Multiple Myeloma and other Myeloproliferative disorders are known to exhibit high levels of Hb F and therefore HPFH, though the exact mechanism of how this is achieved is not completely understood. Hb F profiles of patients suffering with haematological malignancies and what therapy is taken for their disease are being carried out. The correlation between disease, drug type and Hb F level will be determined for each patient and test whether this same therapy is responsible for the elevated levels of Hb F and to what extent. The results from this study are expected to provide additional information on how Hb F responds to external compounds and drugs. Finding a way to elevate high levels of Hb F in patients suffering from β thalassaemia major would be of great benefit and help treat their symptoms. This will enable the possibility to render them transfusion independent and improve the patient’s medical and social life.

References
Giardine, B., and Borg, J.et al., (2011) Systematic documentation and analysis of genetic variation associated with hemoglobinopathies using the microattribution approach. Nature Genetics. 43(4):295-301

In collaboration with the Laboratory of Molecular Genetics, Department of Physiology and Biochemistry

Important clinical models such as people with hereditary persistence of foetal haemoglobin (HPFH) or others such as carriers of foetal/adult haemoglobin variants (that include Hb F Malta1 and Hb Valletta) can serve to identify what and how Hb F is regulated and controlled. A large collection of Hb F Malta 1/Hb Valletta heterozygotes are being tested both at the Hb F profiling level, and genetic level. Both cord and adult blood is already available and additional patients are being collected prospectively. Additional patient material from β thalassaemia trait patients are also being identified by testing that is conducted at the Laboratory of Molecular Genetics, University of Malta. It is anticipated that by comparing the different Hb F expression profiles of adult β thalassaemia trait patients as well as the haemoglobin chain profiles of cord Hb F Malta 1/ Hb Valletta vs. the genetic profiles obtained from KLF1, BCL11A and MYB will highlight important features that these genes exert on the globin gene locus itself and to what extent are they modifying the phenotype. Furthermore, adult patients with Hb F Malta 1 / Hb Valletta are tested by real time PCR to quantify the foetal mRNA globin transcripts, and including also a number of resequencing of genes in the large Maltese family that exhibit HPFH. Together this should summarize the role of important genes and their protein products in well-defined models.

Recognising the advantages of the genetic history and composition of the Maltese population, and aiming to excel in this field, in 2012 scientists from the Department of Applied Biomedical Science obtained national R&I funding for establishing High Throughput Sequencing as an area of expertise within the department. The goal of the Malta NGS project is to utilise selected gene panels, exome and whole genome sequencing to identify novel disease-causing genes and mutations within a subset of diseases present in the Maltese population. Maximising the potential of this technology, and further strengthening the close ties that exist locally between scientists and clinicians, this project is a collaborative effort between scientists and clinical experts, thus employing the expertise of specialist clinicians in the identification and phenotyping of patients and the technical expertise of scientists in setting-up the collections and carrying out laboratory analyses. This project includes research on various conditions including, amongst others, hypertrophic obstructive cardiomyopathy, polycystic kidney disease, familial hypercholesterolaemia, myocardial infarction, obesity, Paget’s disease of bone, pseudoexfoliative glaucoma, speech language impairment, Hirschsprung disease, Idiopathic hypogonadotropic hypogonadism and autism spectrum disorders. Additionally, the NGS project is serving to train a number of MSc and PhD students in this field. The vast amount of data which is being generated by this project will require extensive bioinformatics analysis within the context of the specific disease, lifestyle and environmental influences which are also being captured during patient recruitment.

References

T. Felice, R. Attard, F. Borg Carabott, R.G. Xuereb, K. Cassar, R. Farrugia, S. Bezzina Wettinger. Phenotype-genotype correlation in familial hypertrophic cardiomyopathy in the Maltese population; MMJ 2018; Volume 30 Supplement: P4.22 p137

Muscat Y, Camilleri G, Borg Carbott F, Cassar K, Mallia M, Vella N, Bezzina Wettinger S, Farrugia R. High Throughput Sequencing identifies PINK1 p.G47R: A rare mutation identified in a Parkinson’s disease patient;  Eur J Hum Genet; 27, 870–1041 (2019) E-P09.33

G. Camilleri, S. Camilleri, S. Bezzina Wettinger, R. Farrugia. A novel mutation in LRRK2 influences risk for Parkinson disease in the Maltese Population; Eur J Hum Genet; 2015: Vol 23 S1 p202

Pleven A, Said-Conti V, Borg Carbott F, Attard R, Cassar K, Bezzina Wettinger S, Farrugia R. ADPKD mutation in a small island population.  Pediatr Nephrol; 2017: 32 p1765

A. Pleven, V. Said Conti, F. Borg Carbott, R. Attard, K. Cassar, S. Bezzina Wettinger, R. Farrugia. High throughput sequencing identifies p.Y1435X as a novel truncating PKD1 mutation.  Eur J Hum Genet; 26, 820–1023 (2019) E-P03.31

Dingli P, Attard R, Cassar K, Farrugia R, Bezzina Wettinger S. Next Generation Sequencing of Familial Hypercholesterolaemia-related Genes in a Mediterranean European Cohort; Eur J Hum Genet; 2016: Vol 24 S1 p124

Dr Stephanie Bezzina Wettinger from the Departmentt of Applied Biomedical Science, Faculty of Health Sciences, University of Malta, has received a Research Excellence Award from the University of Malta to use high throughput sequencing to identify genes underlying Pseudoexfoliative Glaucoma, a common cause of visual impairment and blindness. Together with her collaborators Dr Rosienne Farrugia, Ms Francesca Borg Carbott, Dr Jean Paul Ebejer, Dr Karen Cassar and Mr Francis Carbonaro they will apply these findings to develop tests to identify people at risk and new drug targets, and to determine associations with myocardial infarction since literature suggests an association of glaucoma with cardiovascular disease.

 Autism spectrum disorder (ASD) is a neurodevelopmental condition that affects the social interaction, verbal abilities and behaviour of one in every 68 children in the USA alone. In Malta, statistics are not yet available, but ASD is not that uncommon.

 Now, as part of the Malta Next Generation Sequencing (NGS) Project, we are trying to unravel the genetic causes of ASD.

 What is particularly intriguing about the study is that Dr Bezzina Wettinger and her team plan to recruit families with more than one affected individual to be able to understand how genetic variant play a role in these types of conditions.

 https://researchtrustmalta.eu/blog/the-genes-behind-autism/