Projects

Research Activities

The initial aim of the Center is the development of research projects in the field of inherited kidney disorders. In the future and depending on funding, there will be expansion in the direction of other clinical entities, in collaboration with other colleagues. The material collected and archived in the Biobank is typically DNA (from peripheral blood, or saliva, or other sources), plasma, serum, urine and a biopsy (renal or other, when available), along with demographic and medical information of the participating volunteers. All participants are informed by one of the medical doctors or other qualified research personnel and sign an informed consent form which has been approved by the Cyprus National Bioethics Committee (document EEBK3). All medical records and biological material are kept under secure conditions at a specially prepared Biobank space with absolute confidentiality and protection of sensitive data of personal character. Access to the data requires a password which is provided by the Coordinator to authorized personnel and collaborators. Similarly, access to the biological material is under the approval of the Coordinator and the Academic Council of the Research Center.

There are several research directions in progress, combining molecular and cell biology approaches, clinical and molecular genetics, while a major research activity pertains to the characterization of a mouse model for Alport Syndrome. A general overarching aim is the understanding of the molecular and cellular processes that cause disease, thereby hoping to achieve better diagnosis, prevention and therapy of disease.

Projects in progress are the following:

Clinical and molecular investigation of familial microscopic hematuria of glomerular origin
1. Nephropathies of Collagen Type IV
2. C3 Glomerulopathy (CFHR5 nephropathy)
Search for genetic modifiers that predispose patients with thin basement membrane nephropathy to focal segmental glomerulosclerosis and renal failure, on long follow-up. Also, follow-up of patients with CFHR5 nephropathy with the aim to search for genetic modifiers.
Follow up of patients with MUC1 kidney disease with the aim to search for early biomarkers and genetic modifiers
Investigation of the potential role of mir-548c-5p as a regulator of FOXC2 transcription
Follow-up and characterization of a knock-in mouse model for Alport Syndrome, carrying mutation Col4a3-p.G1332E
Adolescent psychopathology cohort (this is a project developed and coordinated By Dr Kostas Fanti, Assistant Professor at the Department of Psychology of the University of Cyprus)

Collaborating Centers / Partners

Laboratory of Molecular and Medical Genetics, University of Cyprus
Departments of Nephrology of the Public General Hospitals in Nicosia, Limassol, Larnaca, Paphos, Famagusta
Department of Nephrology, Hippocrateon Hospital, Nicosia
Department of Histopathology, Nicosia General Hospital
Department of Pediatric Nephrology, Archbishop Makarios III Hospital, Nicosia, Cyprus
Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
Departments of Nephrology at Medical School of Aristotle University of Thessaloniki; Medical School of University of Crete; School of Medicine, University of Patras; Medical School, University of Athens
University College London-Centre for Nephrology, London, UK
University of Heidelberg, Medical Research Center, Mannheim, Germany
Clinic of Nephrology and Rheumatology, University Medicine Goettingen, Goettingen, Germany
Harvard Medical School and Broad Institute of Harvard & MIT, Cambridge, Massachusetts, USA
Wake Forest School of Medicine, Winston-Salem, North Carolina, USA
Centre for Advance Studies, Research and Development in Sardinia (CRS4), Italy

Brief description of projects

1. Clinical and molecular investigation of familial microscopic hematuria of glomerular origin

a) Nephropathies of Collagen Type IV

This project aims at archiving and investigating families with patients that segregate a familial form of microscopic hematuria of glomerular origin. There is preference for in depth clinical and molecular examination of families with at least three affected members.

Thus far nearly 300 families from Cyprus and Greece have been archived and have been investigated or are under study. The initial analysis includes the parallel Next Generation Sequencing (NGS) of a panel of five genes, namely COL4A3, COL4A4, COL4A5, CFHR5 and FN1. The first three genes encode the alpha chains of collagen IV, which is the most abundant component of the glomerular basement membrane; CFHR5 encodes the complement factor H related 5 regulator of the alternative pathway of complement (see below); and FN1 encodes the fibronectin. For NGS we use the PGM, Ion Torrent, of Life echnologies. Numerous mutations have been detected in the four genes, excluding FN1 (Papazachariou L et al, 2014).

Mutations in the collagen IV genes result in diseases known as Alport Syndrome, with autosomal or X-linked inheritance and thin basement membrane nephropathy (TBMN) with autosomal dominant inheritance and characteristic histopathology (Deltas C et al, 2015; 2013; 2012). Several founder mutations have been detected, the most frequent of which is a substitution of glutamate for glycine at position 1334 of the α3 chain of type IV collagen (COL4A3-p.G1334E). The mutation has been found in 18 families with 200 patients. It is found in geographic clusters around the island and it originates about 400 years ago. A characteristic feature in this cohort of TBMN patients is the broad phenotypic heterogeneity, which is largely attributed to the role of genetic modifiers (Voskarides K et al, 2007; Pierides A et al, 2009).

b. C3 Glomerulopathy (CFHR5 nephropathy)

This is a heritable form of C3 glomerulopathy, endemic to Cyprus. It was Dr Daniel Gale and collaborators who first diagnosed Greek-Cypriot patients in London, in families that segregated this autosomal dominant condition, with characteristic histology that includes C3 deposition in the glomerulus. The expansion of the studies in Cyprus, using data and material Biobanked at MMRC, revealed 23 families and 175 patients thus far that carry a heterozygous founder duplication of exons 2-3 in the CFHR5 gene. It is interesting that the disease also demonstrates a broad phenotypic heterogeneity whereas men are more frequently affected with a severe symptomatology compared to women, for reasons that remain unknown. The disease is hypothesised to have originated nearly 400 years in the Troodos Mountains, in the Marathasa area (Deltas C et al, 2013).

2. Search for genetic modifiers that predispose patients with thin basement membrane nephropathy to focal segmental glomerulosclerosis and renal failure, on long follow-up. Also, follow-up of patients with CFHR5 nephropathy with the aim to search for genetic modifiers.

A very important observation regarding the clinical presentation of patients with TBMN and CFHR5 nephropathy relates to the broad phenotypic heterogeneity, both between families and within families.

The full spectrum of the phenotype of patients with TBMN (carriers of the autosomal recessive form of Alport Syndrome), behaves as a "multifactorial" condition, implicating primary genes, modifier genes and environmental factors (from Deltas C et al, Nephron 2015)

All patients will develop chronic kidney disease (CKD). However, while many patients with these two conditions will stay for life with microscopic hematuria or low grade proteinuria, others may progress to high grade and even nephrotic range proteinuria and chronic renal failure, even end-stage renal disease (ESRD), usually after the age of 50 years. A substantial percentage of patients with TBMN will develop focal and segmental glomerulosclerosis (FSGS), often times leading to misdiagnosis and inappropriate treatment (Voskarides K et al, 2007; Pierides A et al, 2009;Deltas C et al, 2015; Savva I et al, 2015). These and other studies by several groups, prompted the renal community to agree that the previously used term, "benign familial hematuria", is a misnomer and caution should be exercised in closer follow-up of the patients. There is a clear age-dependent penetrance and in our Greek-Cypriot cohorts, we observe that about 30% of the TBMN and CFHR5 patients develop ESRD by the age of 70 years. We hypothesised that likely genetic modifiers are responsible for this clinical variable expressivity and we are on the search for them, taking advantage of the lesser complexity of our cohorts, with founder mutations. The full spectrum of the phenotype behaves as a multifactorial condition, implicating primary genes, modifier genes and environmental factors (Voskarides K et al, 2012; 2013; Papagregoriou G et al, 2012). At the moment there is in progress the whole exome sequencing of 200 TBMN patients in the hope to identify putative functional DNA variants that may be acting as such modifiers.

3. Follow up of patients with MUC1 kidney disease with the aim to search for early biomarkers and genetic modifiers

Medullary cystic kidney disease (recently renamed MUC1 kidney disease), was diagnosed in several large families in Pafos, south-west of the island, in the 90s. MMRC researchers developed a clinical and genetics project that resulted in mapping the first gene on chromosome 1q21, in 1998 (Stavrou C et al, 1998;2002;Christodoulou K et al, 1998). It took several years before the gene was cloned by a Broad Institute group in the USA (Kirby A et al, 2013; Bleyer AJ et al, 2014). This development revived the interest of researchers and the Broad Institute group proposed a collaboration with MMRC in completing and upgrading the patient registry in Cyprus, which is presently in progress. It turns out that there are about 300 patients followed up in several counties, 150 of which are Greek-Cypriots, archived in our Biobank. Our common interests include the identification of potential early biomarkers, in blood or urine, for timely detection of the patients who run the highest risk to reach ESRD, usually at ages of 50s-70s. The gene is a very complex one and the mutation, a C insertion within a repeated region, is difficult to detect with traditional methods. Therefore, within the framework of this collaboration, all patients, in addition to linkage analysis at MMRC, are diagnosed with a proprietary certified method at Broad Institute.

4. Investigation of the potential role of mir-548c-5p as a regulator of FOXC2 transcription

Podocytes are highly differentiated epithelial cells outlining the glomerular vessels. FOXC2 is a transcription factor essential for inducing podocyte differentiation, development and maturation. It is considered to be the earliest podocyte marker and has been found to regulate the transcription of podocyte related genes. Murine Foxc2 knock-out podocytes fail to differentiate. In addition miRNAs are quite important in podocyte function as well, as podocyte specific dicer knockouts fail to maintain glomerular function. We hypothesize that the transcription of FOXC2 and consequently the differentiation of podocytes, can be influenced by miR-548c-5p through a predicted 21nt-long target region located 8kb upstream the FOXC2 transcription start point.

By using luciferase reporter constructs, it became apparent that the DNA target site acts as a conventional miRNA-binding site. During early differentiation of AB8/13 human podocytes, miR-548c-5p mimics effectively diminished endogenous FOXC2 levels, while a more dynamic model of this interaction was observed when investigating other time points, suggesting a dependence on target site availability. Therefore, the role of this miRNA target site as a distal enhancer/repressor element was investigated using Chromosome Conformation Capture. Evidence indicates a correlation between podocyte differentiation events and the interaction levels between the miRNA target site and the FOXC2 proximal promoter regions. Furthermore, human AB8/13 podocytes were transfected with miR-548c-5p mimics or inhibitors and FOXC2 transcription and translation levels were recorded. In addition, translation levels of WT1, another important podocyte transcription factor, were also determined. Neither of these two genes has a 3'UTR target site for miR-548c-5p. Cellular localization of FOXC2 levels was defined in tested differentiation time points by confocal microscopy.

Collectively, results indicate an important coordination pattern between miR-548c-5p and FOXC2 transcription rates. This study can provide insights on the basic biology in regards to the initiation of podocyte differentiation and expand our understanding in podocyte phenotypes observed in glomerular disease.

5. Follow-up and characterization of a knock-in mouse model for Alport Syndrome, carrying mutation Col4a3-p.G1332E

The mutation carried by the mouse is the recapitulation of the corresponding in human patients, COL4A3-p.G1334E, the most frequent founder mutation among Greek-Cypriots. At the moment 200 patients belonging to 18 families have been archived in our Biobank. Homozygous mice demonstrate activation of the unfolded protein response and have the characteristic ultrastructural pathology of Alport, with alternate thinning and thickening of the glomerular basement membrane on electron microscopy (Pieri M et al, 2014). We are in the process of completing the biochemical and other histological characterization of the mice, which have a rather mild phenotype overall. It is in our plans to use these mice for pre-clinical studies, treating them with synthetic chaperones, in the hope that relieving the ER stress will ameliorate the phenotype.

6. Adolescent psychopathology cohort (this is a project developed and coordinated By Dr Kostas Fanti, Assistant Professor at the Department of Psychology of the University of Cyprus)

Genetic risk for Antisocial Behaviour in adolescence-A Family-Based Association Study: This is a study of adolescents investigating the association of psychopathological symptoms in relation to distinct genetic risk markers. Specifically, saliva samples were collected from 210 adolescents and their parents, from which genomic DNA was extracted. In this sample, a complex disease paradigm is currently being employed to investigate the association between Conduct Disorder and multiple genes that code for a diverse array of neurobiological systems. These at risk adolescents were identified from a larger screening sample of 1,200 adolescents and their families that completed neuropsychological and behavioural measures of psychopathological symptoms, endophenotypes and personality traits.

The aim in current and future work is to identify differential physiological and brain responses to emotional tasks to eventually propose a shift away from traditional diagnosis and a focus on core psychological processes that have a neurophysiological basis. Currently the researchers are studying gene by environment interactions to understand the development of behavioural problems. In addition, they are designing a study to investigate gene by neurophysiology interactions to further understand the development and aetiology of antisocial behaviours.

Relevant Publications: Lordos, A. & Fanti, K. (under review). Executive Dysfunction and Callous Unemotional Traits mediate genetic risk for
Antisocial Behaviour in adolescence: A Family-Based Association Study.

Developmental psychopathology lab: The Developmental Psychopathology lab was established in the Department of Psychology at the University of Cyprus by Dr Kostas A. Fanti. Following the Developmental Psychopathology perspective, the researchers combine questions about developmental change and psychopathology. Their research is primarily focused on investigating the development of different psychopathological problems, such as attention deficit hyperactivity disorder and conduct disorder, from preschool to adolescence.