Projects / Programmes
Genetic basis of erythrocytosis in Slovenia
| Code |
Science |
Field |
Subfield |
| 3.04.00 |
Medical sciences |
Oncology |
|
| Code |
Science |
Field |
| B000 |
Biomedical sciences |
|
| Code |
Science |
Field |
| 3.02 |
Medical and Health Sciences |
Clinical medicine |
erythrocytosis, congenital, diagnostic algoritm, genetic diagnosis, NGS, genetic variants, functional analysis, disease model
Researchers (24)
Organisations (4)
Abstract
Within the project Genetic basis of erythrocytosis in Slovenia (GenEry) we aim to addresses current unmet diagnostic needs for a rare haematological condition, congenital erythrocytosis. More than 60% of patients in EU are left underdiagnosed, moreover diagnosis in Slovenia is not yet established.
Erythrocytosis is a haematological condition with increased mass of erythrocytes and consequently increased haematocrit and haemoglobin. It is caused by inherited or acquired mutations or as a compensatory mechanism in some chronic diseases (heart, lung, kidney, etc). Clinical signs are often overlooked until complications; e.g. thrombosis that could result in mortality. Therefore rapid and accurate diagnosis is of high importance.
The genetic cause of erythrocytosis may be polycythemia vera or congenital (familial) erythrocytosis. Polycythemia vera is the most common acquired erythrocytosis and JAK2 mutations are routinely diagnosed, also in Slovenia. Congenital erythrocytosis however has heterogeneous genetic background, making the diagnosis very difficult. Reasons can be mutation in the genes involved in detection of oxygen levels in the blood (EPO, EPOR, VHL, EGLN1, EPAS1) or regulation of haemoglobin affinity to oxygen (HBA1, HBA2, HBB, BPGM, PKLR). Within the European Congenital Erythrocytosis Consortium only 7 out of 21 genes currently associated with the congenital disease are genetically diagnosed using classical but time consuming Sanger sequencing. Due to poor diagnosis treatment is not well defined and medical doctors do not share a common opinion regarding best treatment selection (low dose aspirin, venesection, etc.).
Within the project we will apply holistic, NGS diagnostic solutions in aim to update diagnostic algorithm and focus on discovering new disease mechanism. Development of NGS for the diagnosis of congenital erythrocytosis will enable faster and cheaper analysis of patients and simultaneous analysis of all target regions/genes of interest in one test. Project is imply 5 working packages focusing on diagnostic as well as clinical aspect of disease. Within WP1 we will evaluate etiological data of disease in Slovenian population and update current diagnostic algorithm. WP2 will focus on development of classical Sanger sequencing as well as new NGS diagnostic approach. We will analyze genes previously associated with the disease as well as new genes, including exonic, intronic and regulatory regions. WP3 is designed to understand functional impact and molecular mechanism of newly discovered genetic variants, subsequently in updated disease model. Within the WP4 we will design and validate NGS panel for research of idiopathic erythrocytosis. Furthermore, we will plan to develop additional simple and quick genetic tests for verification of most commonly discovered genetic variants. The focus of WP5 is management and disseminatioion.
Improved, quick and cheaper diagnosis developed within our project will unable to better understand disease mechanism and consequently patient stratification. Thereafter the best treatment practice to each patient subgroup will be possible (personalized medicine).
Significance for science
Improved clinical characterization. Based on our own experience and literature review we will upgrade current diagnostic algorithm for erythrocytosis (PV), revealing the need for additional diagnostic procedures and genetic tests. Diagnostic algorithm for congenital erythrocytosis in Slovenia will be established for the first time.
Improved diagnosis will enable to offer best treatment practice to each patients subgroup (personalized medicine). Patients will be followed up by one specialist. In some cases no treatment is required and condition is not indicated as disease.
Development of new Diagnostic methods. We will establish genetic diagnostic with Sanger sequencing for all genes currently causative for congenital erythrocytosis and transfer it to the routine practice at SHL UMCLj-CDH. Slovene patients with idiopathic erythrocytosis will thereafter have the possibility to obtain specific diagnosis and receive the best personalized treatment, currently impossible with the available diagnostics.
Even if Sanger diagnostic in EU is routinely in used, more that 60% of patients still remains unidentified. This indicated the need for further development of new diagnostic test in this field. We plan to develop quick and simple genetic test for most commonly found genetic variants.
In parallel genetic diagnosis with next generation sequencing (NGS) will be developed and transferred to routine practice at SHL at UMCLj-CDH. NGS will enable faster, cheaper and holistic diagnosis and could aid in identification of new regions associated with the disease. The knowledge will facilitate the development or translation of future diagnostic methods to NGS.
Development of In-house Basic Research. Scientific findings described above belong to the category of fundamental scientific findings in the field of medicine and genetics. Beside clinical part we will also perform basic research. Functional analysis of newly discovered variations will enable us to understand the disease mechanism, its molecular background and to prepare revised models of the disease mechanisms. Therewith we will further develop expertise in the field of functional genomics, biochemistry and molecular biology.
This altogether represents a venue towards translational medicine where genetic methods are used to understand disease background and mechanism, leading to improved human health.
Development of Other Fundamental Sciences and New Technologies. Project will further development of molecular medicine and associated research fields in the post-genome era. The state-of-the-art diagnostic and functional genomics tools will be applied, in line with international guidelines. In addition to offering fundamental knowledge in the basic fields of medicine, genetics and molecular biology, the project also reaches into the domains of bioinformatics and modeling. It is expected that investigations planned within the proposed project will exert an influence on all stated fields.
Significance for the country
The project will apply a combination of clinical, experimental and bioinformatics approaches that can significantly contribute to cost reduction and to the increased quality of health-care. Wide availability of various experimental data and biomedical knowledge resources requires close collaboration between health care providers, researchers and modelers.
Upgraded clinical characterization of patients and new diagnostic methods will enable quick diagnosis of currently under-diagnosed patients. Consequently patients will not be sent to several specialists and will not have to endure several unnecessary special diagnostic procedures, reducing the workload for medical doctors, diagnostic laboratories and absence from work for patients. All together this will result in reduced health insurance costs. Project will contribute to the state-of-the-art trends in translational medicine as well as to “health literacy” of citizens.
The development of NGS sequencing is particularly important for diagnosis of rare diseases, where many regions are involved in disease phenotype. NGS analysis is quick, economic and needs small amounts of biological samples. Sequencing with NGS technology allows scientists to explore encoding and regulatory regions in the genome, identify variants for population studies, genetic diseases and tumor research with much greater accuracy and considerably lower costs compared to sequencing according to the Sanger method. Such research will be extremely important in the future. Individuals will, among other things, be able to identify a predisposition to the development of certain diseases, thereby reducing the costs of treatment.
At the UMCLj, the cost for Sanger analysis of one-fold coverage of 450 bp sequence is approximately 150 €. If we want to sequence coding and regulatory regions of several genes (20 genes = ~40.000 bp), several successive Sanger sequencing steps are necessary, rendering the procedure very lengthy and cost consuming. With the NGS sequencing, the region of 40,000 bp can be sequenced in one step with more than 500 times the coverage within the cost range of one Sanger sequence (Table 2).
The proposed project will contribute to the focused applicative research in the field of molecular diagnostics. During the project we will develop NGS panel and NGS pipeline for research of erythrocytosis. Acquired expertise and know-how will be applied to the future fields of clinical research and diagnosis. With this we will open the avenue for the small and medium enterprises, which are the most interested industries for further development and marketing.
Table 2. Comparison of Sanger and NGS sequencing strategy.
Sanger (1 gene)
Sanger (20 genes)
NGS (20 gene)
Sequencing region
450 bp
40.000 bp / 450 bp =
~89 runs
40.000 bp
Coverage
1 x
1 x
500 x
Cost per sample
150 €
150 € x 89 =
~13.350 €
150 €
Most important scientific results
Interim report
Most important socioeconomically and culturally relevant results
Interim report
