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INTEGRATING ARMENIA INTO ERA

Final Report Summary - INARMERA (INTEGRATING ARMENIA INTO ERA)

Executive Summary:
Integration of the South Caucasus countries into European Research Area is one of the priorities of EU’s European Neighborhood Policy. This is the objective of ERAWIDE initiative aimed at reinforcing the cooperation capacity of a prominent R&D center in these countries by providing the possibilities to improve its research activities in the areas of thematic priorities of FP7. The government of the Republic of Armenia recommended as such a center the Center of Medical Genetics and Primary Health Care (CMG) due to its high reputation, broad international contacts, participation in FP7, and the importance of the top-level research in Medical Genetics for general population. To reach this strategic objective, the INARMERA project consortium was working on three parallel directions: educational, research, and networking for the development of the strategy and recommendations addressed to the Armenian and the EU authorities.

INARMERA consortium members developed special training modules and performed the professional training of the CMG scientists in preparation of EU FP7 projects and in latest professional advancements of medical genetics through dedicated training sessions in different European research centers. CMG specialists have reached four significant achievements for education and practice in medical genetics: higher professionalism, broader medical knowledge, practice-based improvement, and better communication skills.

Special guidelines were developed which helped to initiate four pilot research trials in the CMG supported by European participant institutions which were successfully finalized by the end of the project. The performance and the analysis of the pilot trials recorded the progress the CMG scientists have reached in the planning, implementation, and analysis of the research trials. The pilot trials were served as an initial platform for the creation of new strong and mutual research collaboration and development of new research project proposals.

For networking and dissemination purposes a special plan was developed which helped to present the activities of the CMG and INARMERA project in different Armenian and European events as well as set up joint EU-Armenia research consortiums and develop new project proposals.

Finally, a Strategy Expert Group was set up involving both Armenian and European specialists who performed the research assessment of the CMG. The evaluation of the research potential of the CMG helped to develop the Strategy for the CMG to improve its research and health care capacity, enlarge its regional scale, respond to socio-economic need of Armenia, as well as turn it into a reliable partner for European institutions in EU Programs.
Project Context and Objectives:
The problem INARMERA addressed was the not fully exploitation or missed opportunity of strong S&T cooperation between Europe and Armenia in the mutually important field of science and technology, and the benefits it could bring to both sides.
INARMERA project proposed several dedicated activities aimed to improve the cooperation capacity of Armenia which could complement the Research and Innovation activities described in the Armenian National Indicative Program covered by the European Neighborhood and Partnership Instruments (ENPI).

The goal of INARMERA project was the realization of the following improvements in EU-Armenia cooperation in Framework Programs, involving the Center of Medical Genetics and Primary Health Care (CMG) in Armenia:
• Enhanced quality of cooperation - more frequent contacts and meetings and different levels of cooperation;
• Enlarged scope of cooperation - more EU countries involved, more research centers and businesses involved, and more topics of mutual interest covered;
• Deepened content - more joint research activities;
• Increased number of FP7 project proposals with participation of Armenia;
• Strengthened links between policy makers in the EU and Armenia in science and technology.

The Strategic Objective of the INARMERA was to reinforce the cooperation capacity of the CMG in Armenia by creating the possibilities to improve its research activities in the areas of thematic priorities of FP7. The Participants of INARMERA have identified three clearly defined Specific Objectives that guided the project participants during the lifetime of the project towards the achievement of the project goals and helped to reach the Strategic Project Objective while keeping the later constantly tuned to the Objective of the FP7-INCO-2010-6 call. The overall aim of reinforcing the cooperation capacity of the CMG was decided to achieve by implementing support actions on three levels, each of them corresponding to each specific project objective:

1. Educational level: deploy a set of supporting activities to enable development of training modules, sessions in Armenia as well as professional training of the CMG researchers in Europe to build competency and facilitate the participation of the CMG in Framework Programs.

2. Research level: deploy a set of supporting activities to intensify contacts between the CMG and the research organizations in the Members States and Associated Countries to provide means for networking through participation of CMG researchers at different events in Europe and workshops organized in Armenia, and means for networking and dissemination campaigns in Europe and Armenia to enable new collaborations leading to new R&D projects in the fields of mutual interest. The CMG and their EU partners also had come up with four pilot trials in Medical genetics for joint management, performance, assessment, and analysis of the research trials of mutual interest and for the final research assessment of the CMG.

3. Analysis and Policy level: deploy a set of supporting activities to enable development of a strategy for the CMG to increase its scale, and in particular its regional coverage, to improve responses to the socio-economic needs of Armenia.

To carry out the most efficient and up-to-date training, new training modules were prepared. EUROTEX organized six training sessions devoted to polishing the skills, writing better proposals, broadening contacts and networking, and to better understanding of the EU project management. Professional Training in Medical Genetics was organized both in Europe and in Armenia. For this purpose, leading experts from DCS lectured in Armenia for the CMG scientists on the latest achievements and methodologies in Human and Medical Genetics. Another approach of Professional Training was the joint supervision of PhD students by CMG and DCS.

The Participants developed Networking and Dissemination Plan to guide CMG in its networking with research centers in Europe and in Armenia. EU Project Unit was set up consisting of active CMG researchers constantly monitoring new EU calls for proposals and through successful networking establishing project consortia and preparing and submitting project proposals.

The Participants developed guidelines and carried out four Pilot Trial Projects with partners in Europe:
• Study of the genetic structure of Armenian population in connection with other European populations in the context of disease association studies:
• The prognostic value of S100 proteins in Familial Mediterranean Fever
• Genetic attributable risk of cardiovascular disease in the Armenian and Georgian populations
• Prediction and assessment of BRCA1 and BRCA2 variants of unknown significance

The Participants jointly analyzed the outcome of all Pilot Trial Projects, following the instructions in the Guidelines. Two interim (months 18 and 36) and one final status reports (month 36) with the analysis of the results of the Pilot Projects were prepared which were taken into account for the development of the Strategy of the CMG.

The Strategy Experts Group (SEG) was set up and held three meetings and three site-visits to validate and supplement the content of the self-evaluation report to test the preliminary research assessment of CMG. After discussion and consultation on the updated documentation of the CMG regarding the terms of reference set as the criteria for its research assessment, the SEG developed and delivered the final version of the Research Assessment of the CMG (month 34). After finalizing the research assessment of the CMG, the SEG moved to the next stage - development of the Strategy. The Coordinator CMG was responsible for the composition and activities of the SEG. The Strategy was updated (month 24) given the inevitable positive development at the CMG in course of INARMERA. After taking into consideration the remarks by the external experts, the final Strategy document was prepared (month 36). In its final task INARMERA consortium summarized three-year long experience and drew recommendation on R&D cooperation with Armenia and the ways to increase its efficiency.
Project Results:
Professional Training
Professional training of CMG specialists was organized in the form of workshops in Armenia and short- or mid-term trainings in DCS or other European research centers under the supervision of European researchers. After the revision of the Research Assessment of the CMG and identification of its needs, the professional training plan was composed, involving the Next generation sequencing (NGS) technologies in practice and in clinic, prenatal diagnosis, and biobanking and networking in Europe. These targeted training topics allowed to train specialists required for the research activities of the CMG, to overcome new technological/methodological barriers in the field of human genetics, and to gain experience and establish new contacts with European experts.

In total six professional training sessions-workshops were organized in line with one of four Research Priority Areas (RPA) of the CMG by participation of 10 specialists from four European centers (University of Copenhagen, Danish Cancer Society - Denmark, Institute of Myology - France, and Children’s Hospital & Institute of Medical Genetics - Switzerland). The 1st Professional Training Session in Genomic Medicine and Clinical Genetics Research as a RPA of CMG was organized by DCS and saw the participation of 34 students from all over Armenia. The 2nd Professional Training Session in Bioinformatics as the RPA of CMG saw the participation of 14 scientists from all over Armenia. A specific session was dedicated to a thorough overview of specialized molecular techniques, DNA sequencing technology, molecular diagnostics with Next Generation Sequencing and its bioinformatics analysis. The 3rd Professional Training Session in line with CMG RPA of Health services for genetic diseases saw the participation of 47 researchers and physicians from all over Armenia and was devoted to the genetic counseling and semiology of neuromuscular disorders. The 4th Professional Training Session with participation of more than 50 researchers and physicians from all over Armenia included a round-table discussion of clinical aspects of diagnostics of dismorphic syndromes and a workshop dedicated to the clinical-genetic counseling of patients with metabolic syndromes. The aim of the last fifth and sixth Professional Training Sessions was the introduction and practice on emerging new immunological methods and nanoparticles, respectively, with research and practical applications in Medical Genetics which brought the participation of 20 specialists from different Armenian research institutes.

In the frame of the second pilot trial project (The prognostic value of S100 proteins in Familial Mediterranean Fever) of the INARMERA project, the consortium initiated a joint supervision of one PhD program at CMG by CMG and DCS senior specialists. The successful progress of the PhD program resulted in publication of four scientific articles and preparation of the thesis which defense is scheduled on March 2014.

Given the rapidly developing field of human and medical genetics, the INARMERA consortium provided more possibilities for CMG specialist to be trained in different European centers. Six short or mid-term trainings were organized for seven CMG researchers in different European research centers:
1. Hospital Henri Mondor (Creteil, France): up-to-date technologies and their applications in molecular genetics diagnostics, as well as the set up a workflow for the screening of DNA sequence variants and their further clinical classification in collaboration with French colleagues;

2. European Society of Human Genetics Conferences in 2011 (Amsterdam, the Netherlands) and 2012 (Nurnberg, Germany): diagnostic applications of next generation sequencing, prenatal diagnostics, array-CGH in cytogenetic lab;

3. Institute of Myology (Paris, France): the counseling of patients with neuromuscular disorders;

4. 15th Summer School of Myology (Paris, France)

5. European Society of Human Genetics Conference in 2013 (Paris, France) provided several workshops and education sessions, including i) IntegraGen workshop on valuable exome and transcriptome sequencing needs to integrate robust processes; ii) CARTAGENIA workshop on next generation sequencing and arrays in postnatal and prenatal clinical practice; iii) Human Genome Variation Society’s workshop on clinical applications of Next Generation Sequencing; iv) Educational session of prenatal and preimplantation genetic screening aimed on integration of microarray technology into prenatal diagnosis; and v) educational session of cancer risk in developmental syndromes.

6. Department of Cellular and Molecular Medicine of the University of Copenhagen, Denmark: Ion Torrent Technology and its applications, transition to the clinic, identification of causative mutations for rare genetic disorders through whole-genome, targeted genome or mRNA resequencing, the application of NGS for family genomics, and NGS data analysis tools. In the scope of this training, a special workshop on NGS analysis presentation was organized in collaboration with Center for Genomic Medicine (Rigshospitalet, Copenhagen, Denmark).

Summarizing the three-year professional training, CMG specialists have reached four significant achievements as a set of core competences for education and practice in genetics health care and research in medical genetics: higher professionalism by demonstration of professional responsibilities and adherence to ethical principles, broader medical knowledge by demonstration of knowledge about established and evolving biomedical sciences, practice-based improvement by the ability to evaluate and assimilate scientific evidence to improve practice, and better communication skills through effective information exchange and teaming with research collaborators. As a result of the core competences gained during the professional training, the CMG specialists currently stand on a higher level of experience and ability to supervise and direct the operations of a genetics laboratory, including all technical and quality assessment procedures, have a broad knowledge of medical genetics and understand the etiology, pathogenesis, clinical manifestations and management of human genetic disorders, participate in research and in the introduction of new methods, apply and interpret whole genome or exome sequencing, apply genetic knowledge to formulate, design, implement, and monitor a research project; assess and participate in a translational research, and provide results, interpretation, and follow up recommendations to professionals.


Setting up EU project proposals
EUROTEX and DCS prepared the training modules. Three training modules and sessions were organized by EUROTEX in Armenia by participation of more than 20 CMG employees. During the first training session the Armenian specialists were trained i) on the structure of FP7, preparation and submission of proposals, and aspects of the ICT Program. The second training session was devoted to the Horizon 2020, and the third one was devoted to polishing the skills, writing better proposals, broadening contacts and networking, and to better understanding of the EU project management. As a result of three consecutive training modules, the Armenian specialists have gained better understanding of the 7th Framework Program and Horizon 2020, and their structures, the rules for participation in and management of the EU projects, financial regulations and reporting.
One of the indicators of successful networking of INARMERA was the preparation and submission of new EU project proposals. The consortium set up EU Project Unit consisting of four active researchers from the CMG and one representative from EUROTEX and DCS. The activity of the CMG researchers of this unit besides successful networking was the establishing of consortia, development and monitoring of project proposals, and their final submission to corresponding agencies. The role of representatives from EUROTEX and DCS was mainly providing information on new EU calls and monitoring the process of submission. Most importantly and as the top of the trainings, four separate proposals were developed and successfully submitted by the Armenian specialists:

1. “Impact of moderate- and high-risk DNA sequence variants in breast cancer risk and relevance to anti-cancer therapy” was prepared and submitted to International Science and Technology Center 2012. The work proposed overlaps with problems of genetics of complex traits and mechanism of oncogenesis of hereditary breast cancer and molecular characterization of BRCA1/2 breast tumors. The consortium of this project proposal consists of 6 partners: the CMG (as coordinator), Institute Curie (Paris, France), University of Montreal (Montreal, Canada), Danish Cancer Society Research Center (Copenhagen, Denmark), Asper Biotech (Tartu, Estonia), and University Paris Descartes (Paris, France). All foreign collaborators have thoroughly reviewed the proposal and gave their interest and commitment to participate in this project which overlaps with their research activity and interests.

2. “Research of biomarkers for disease diagnosis, disease monitoring and therapeutic treatment response in Duchenne Muscular Dystrophy patients (IBiSD study)” multicenter proposal was set up between the Institute of Myology, Paris, France, the Yerevan State Medical University and CMG. The aim of this project is to identify biomarkers for the diagnosis and monitoring of Duchenne Muscular Dystrophy (DMD) from a transcriptomic, proteomic and inflammatory point of view. The project involves the creation of a cohort of children with DMD and their age-paired and ethnic origin-paired controls, from whom blood and urine samples will be taken.

3. “Reinforcing Cooperation with Eastern Partnership Countries on Bridging the Gap Between Health Research and Innovation” proposal development was initiated by communicating with several medical genetics centers in Eastern European Partnership Countries and innovation support centers and industrial SMEs from the EU, and setting up of the consortium involving CMG, Institute of Genetics and Cytology, Belarus, Tbilisi Cancer Centre, Georgia, National Cancer Institute of Ukraine, Ukraine, European Centre for Knowledge and Technology Transfer, Belgium, Lithuanian Innovation Centre, Lithuania, Asper Biotech, Estonia, and ViennaLab Diagnostics, Austria. This strategic goal was the implementation of actions to support and promote knowledge transfer projects, including four case studies originated in participant organizations from EaP countries; means for organization of workshops and brokerage events, networking for pooling knowledge and identifying tangible opportunities; for exchange of scientists and training in knowledge and technology transfer including development of business models and pilot services. R2I-HEALTH proposal submitted to the FP7-INCO-2013-9 - EC FP7 specific program Cooperation call, allowed direct participation of several CMG specialists in its development, improving skills in contacting, networking, assessing the research potential, as well as in other aspects of preparation, writing and submitting the EU project proposal.

4. “Developing Medical Genetics Education through Curriculum Reforms and Establishment of Postgraduate Training Programs (MedGen)” proposal was submitted to the “Curricular Reform” - 6th call of the EU TEMPUS IV program of the Directorate General for Development and Co-operation. The wider objective of MedGen was the development and implementation of solid basis with active workforce of Medical Genetics corresponding to EU recommendations of educational and professional standards and reinforcement of international cooperation capacity in Armenia and Israel (cross-regional project). Acting as a project coordinator, the CMG negotiated and involved Russian-Armenian (Slav) University and two Israeli universities: Tel Aviv University and Technion-Israel Institute of Technology. To establish harmonized education in MG and to establish a hub for international education and research programs, the following EU higher education universities were involved in the project consortium: Alma Mater Studiorum Università di Bologna, Italy, University of Plymouth, UK, Charles University in Prague, Czech Republic, Medical University of Graz, Austria, and Université Paris Descartes, France. Importantly, all specialists of the CMG who also teach Medical Genetics were involved in the preparation and development of the project.

Development of Networking and Dissemination Plan
The development of the Networking and Dissemination Plan was in line with the strategic objective of the INARMERA to reinforce the cooperation capacity of the CMG by creating possibilities to improve its research activities in the areas of thematic priorities of FP7. The Plan was reviewed and updated by all participants of the INARMERA consortium. To achieve the goals of the Networking and Dissemination Plan several support actions have been developed and deployed corresponding one of the specific objectives of the INARMERA at research and health care levels which consist the core of INAMERA networking and dissemination interconnected strategy. The plan of networking and dissemination included networking and awareness rising activities, dissemination of information about collaborative opportunities, created by INARMERA for scientists and business people, as well as dissemination of INARMERA results to general public both in Armenia and in Europe. The realization of the Networking and Dissemination Plan was performed with the employment of different means: creation of the INARMERA project website, realization of the project leaflets, promotional and information materials, and dissemination amongst EU and Armenian R&D organizations, policy makers, medical and human genetics societies and associations; activities through building cooperation and creating synergies with national and EU projects; presentations, meetings between EU and Armenian scientists through participation in international events and conferences in Europe; and platforms for discussions and setting up of new projects.


Pilot Trial Projects
The definition of criteria and characteristics of INARMERA Pilot Trial Projects (PP) were the initial activities as well as the organization of web-meetings with European research partners collaborating in the frames of the PPs to develop such criteria. The Guidelines for Pilot Trials were prepared for successful start, implementation, monitoring, analysis and eventual comparison and recommendation of PPs. In brief, they describe start and accomplishment of a high value R&D PPs in Medical Genetics between EU and Armenian scientists, administration of potential questions regarding Intellectual Property Rights, ability of cooperation in the frame of a EU or Armenian R&D Project and generation of new knowledge, joint publications, and eventually possibility of continuation in the form of new EU or bilateral R&D projects.
Four approved PPs, involving CMG and at least one participant organization from EU country were started since the beginning of the project and were finalized and analyzed by the end of the project. Every group of CMG researchers involved in each individual PP organized internal meetings and web-meetings with European participant(s) to draw the workplan of PPs according to the criteria and characteristics described in the guidelines.

PP1. Study of the genetic structure of Armenian population in connection with other European populations in the context of disease association studies (in collaboration with INSERM U781ildren, France, National Center of Genotyping, France, INSERM U946, France)
The aim of the PP1 was the study the genetic structure of the Armenian population in connection with other European populations with applications to disease association studies. First, “Genealogy and ancestry information form” was developed with informed consent form which have been obtained for each of the study participant before any blood collection qualified for use both in Armenia. Six target regions were identified, and CMG researchers organized 6 visits to those regions and collected blood samples from 188 individuals having ancestors from the present-day Republic of Armenia and Historical Armenia. DNA was purified from blood samples, and DNA quantity and quality was checked, and genotyping was performed by Illumina Human 660-Quad, consisting of genome-wide distributed SNP markers. The genotyping of selected cases was performed in two stages: genotyping of individuals whose 4 grand-parental ancestors were coming from single region, and genotyping of individuals whose 4 grand-parental ancestors were coming from two different places. This distinction provided possibility to analyze the whole-genome SNP-profile of individuals with one geographic region of ancestors and then compare the genotyping data received from the second group with the same data of the first group. The raw data of more than 600,000 SNPs of each studied individual were analyzed by different computational programs. With Plink software were performed quality control statistics, population stratification detection, and basic association testing. Using Eigensoft software heterogeneity, existence of sub-groups and genetic structure of each studied population were estimated which was compared with the data of other studied European populations. Through principal component (PC) analysis the genetic map of Armenian population as well as of Europe were constructed highlighting existing intra-population differences. The EuroAIMs was identified from the original panel of Europeans by defining northern and southeastern cohorts of individuals on the basis of extreme polar values in PC1. Unbiased FST was then calculated for each SNP as a measure of genetic distance between the two groups. All SNPs were ranked by FST, with those showing the highest values likely to represent the best northern-south-eastern EuroAIMs. The northern cohort included all Finnish and Polish, most German, Irish, and English, as well as some Basque and Italian individuals. All Armenian, Jewish, Greek subjects, as well as other Italian samples were included in the south-eastern cohort. Surprisingly, despite Armenians are included in the south-eastern cohort, they are represented by a single and isolated regional autochthon cluster with low level of subgroup diversity which is mostly explained by relative isolation from other ethnic groups. Currently, a one manuscript is developing to publish in a peer-reviewed journal.

PP2. The prognostic value of S100 proteins in Familial Mediterranean Fever (in collaboration with Institute of Cancer Biology, Denmark)
The central question of the PP2 was the relevance of S100 proteins to autoinflammatory state in Familial Mediterranean Fever (FMF). Samples were collected from a total of 99 patients. 25 healthy population controls were selected age-matched with recruited patients. Plasma samples were categorized into three groups according to the clinical activity of FMF, between attacks, during attacks and in patients with renal amyloidosis. Samples were analyzed using (i) S100A4-specific sandwich ELISA developed in DCS using S100A4-specific poly- and monoclonal antibodies raised in DCS; and (ii) Western blot analysis of plasma samples for correlation of S100A4 and SAA levels and their conformational forms. Level of S100A4 has been determined in three pilot experiments/plasma collections including patients at various clinical activity stages of FMF and controls without clinical signs of FMF. First the levels of S100A4 in plasma of FMF patients at different activity phases of FMF were analyzed. First, we found a tendency correlating genotype of FMF patients with the increased levels of S100A4 in FMF patients’ plasma. Levels of S100A4 and SAA in FMF patients’ plasma were checked at various phases of the disease, between (S) and during attacks (SA). The data revealed an interesting correlation between high levels of SAA and detection of modified extra band of S100A4. We assume that existence of such modified forms of S100A4 might be responsible for its biological activity and can contribute to the pathogenesis of FMF. Analysis of the correlation of the levels of S100A4 and SAA with a genetic background of FMF patient and clinical pattern of the disease found that i) MEFV mutation homozygosity is associated with high probability (100%) with high plasma levels of both S100A4 and SAA and i) patients with low levels of both S100A4 and SAA show no signs of arthritis and myalgia. Therefore, we could conclude that S100A4 concentration is significantly high in plasma of FMF patients compared to healthy persons, and specific conformational forms of S100A4 also might have significance in the pathogenesis of FMF and particularly in its deadly complication, AA amyloidosis. S100A4 might be a valuable biomarker for monitoring FMF activity. It might even sense subclinical inflammation in patients without febrile attacks. The data obtained in this PP together with previously published observations suggest that S100A4 might play a key role in triggering the inflammatory process and contribute in activation of pro-inflammatory factors such as SAA. Sequentially, SAA proteins produced in various cell types in response to S100A4 would be able to transcriptionally upregulate MMPs, cytokines, and to positively regulate their own expression. The results of the second Pilot Trial have been summarized in five articles published in local and peer-reviewed journals.

PP3. Genetic attributable risk of cardiovascular disease in the Armenian and Georgian populations (in collaboration with Viennalab Diagnostics, Austria)
The aim of the PP3 was to assess the associations of selected haemostatic genetic factors to assess the relative contribution of modest-risk haemostatic gene variants versus potentially high-risk haemostatic gene variants to the genetic attributable risk of cardiovascular disease in the Armenian population. A clinical questionnaire was prepared, and DNA samples were collected from 304 patients diagnosed with different cardiovascular disorders with all clinical data and laboratory test results. 36% and 64% of patients were diagnosed with cardiac and vascular problems. 106 healthy controls without personal or familial history of cardiovascular disorders were recruited. Researchers at the CMG and the ViennLab composed a list of genes to select strong candidate susceptibility genes for case-control mutation screening, and to update the expert opinion based on contemporary data. Nine strong or common mutations and polymorphisms of several genes were selected involved in blood coagulation, regulation of blood pressure, and metabolism of lipids, glucose, homocysteine or iron: FV Leiden (G1691A) and R2 haplotype (H1299R) of blood coagulation Factor V, G20210A of blood coagulation Factor II (prothrombin), C677T and A1298C of 5,10-Methylenetetrahydrofolate Reductase (MTHFR), V34L of blood coagulation Factor XIII, 4G/5G haplotype of plasminogen activator inhibitor-1 (PAI-1), and A3 haplotype (4600A>G) and A1 haplotype (4678G>C) of Endothelial Protein C Receptor (EPCR). Genotyping of selected genetic markers was performed by PCR amplification of individual’s DNA followed by reverse-hybridization assay. A population-based analysis of selected haemostatic genes and their relevance to cardiovascular disease were performed. The per-allele odds ratio (relative risk) of the rare alleles was compared between cases and controls, and calculating odds ratios per unit score by logistic regression. To make some allowance for multiple comparisons, 99% CI were used for individual studies, and 95% CI were reserved for the combined estimates. Genotyping data obtained from patients and controls revealed significant difference of heterozygous (simple) and homozygous carriers of selected markers both between patients and controls, and between patients with cardiac disease (group A) and venous problems (mainly thrombophlebitis) (group B) as well. Overall, there was a association of the 1691A, 20210A, and PAI-14G variants with total coronary heart disease under dominant or recessive genetic models of inheritance. The MTHFR 1298C allele, C1298C genotype, and C677C/C1298C compound heterozygous genotype were associated with overall stroke, and the MTHFR 1298C allele, C1298C genotype, and C677C/C1298C compound heterozygous genotype were strongly associated with ischemic stroke. However, the MTHFR 677T allele, T677T genotype, and T677T/A1298A compound genotype were associated with hemorrhagic stroke. Therefore we could conclude that in the Armenian study population C677T and A1298C polymorphisms of the MTHFR gene are genetic risk factors for hamorrhagic and ischemic stroke respectively, independent of other atherothrombotic risk factors. Analyses of the other factor variants showed no significant overall associations with coronary disease. The study provided first clear indication of moderate and highly significant associations of coronary disease risk with the 1691A variant of the factor V gene and with the 20210A variant of the prothrombin gene for the Armenian population. The findings of our analysis indicated a weakly positive association of the 4G variant of the PAI-1 gene with the risk of coronary disease. We observed also C677T and A1298C polymorphisms of the MTHFR gene as genetic risk factors for hamorrhagic and ischemic stroke, respectively. Detection of some genetic markers only among patients increased their value to be effective biomarkers for cardiovascular disorders. Although our results were consistent with a causal interpretation of the epidemiological data for CVD, they also reinforced the need to undertake much larger studies to assess age-specific and sex-specific associations, any gene-gene interactions, and gene-environment interactions and any modest effects that might realistically be expected in CVD.

PP4. Prediction and assessment of BRCA1 and BRCA2 variants of unknown significance (in collaboration with Hospital Henri Mondor, France, Institute of Cancer Biology, Denmark, International Agency for Research on Cancer, France)
The aim of the PP4 was the assessment of the breast cancer (BC) risk attributed to the high-susceptibility genes for genetically homogenous Armenian population. The pilot trial was focused on early onset and familial BC cases of Armenian ethnicity enriched for genetic predisposition to increase the power of the study. This study was the first to assess the BC risk attributed to the high-susceptibility genes for this genetically homogenous population. DNA samples were collected from a total of 135 patients diagnosed with BC according to the developed questionnaire. 42 cases were with strong familial history of breast or ovarian cancer, 2 cases were diagnosed with breast and ovarian cancer, 12 cases were diagnosed with bilateral breast cancer, 2 male cases with breast cancer, and 77 young cases diagnosed with breast cancer by the age 43 y.o. High-resolution melt analysis (HRM) for BRCA1 and BRCA2 was evaluated on the LightScanner-32 instrument. Sensitivity and the specificity of the method were determined by the analysis of 82 known deleterious controls and a blind screening of 35 patients. The DNA samples tested for BRCA1&2 included 82 variants and 10 wild-type controls, which were all verified by direct sequencing using the Big Dye Terminator method. 51 positive BRCA1 samples scattered over 36 of 40 amplicons were available. 31 positive BRCA2 samples scattered over 31 of 50 amplicons were available. To evaluate the specificity of the method, 15 patients were chosen and blindly screened for BRCA1 and BRCA2 in parallel with direct sequencing of all amplicons. Two panels of 80 and 100 primer pairs were evaluated for HRM analysis of full coding region and splice junction sites of BRCA1 and BRCA2, respectively. The primers were newly designed or derived from current primer sets in use for direct sequencing of both genes. The design of most new primers was performed using the Oligo7 Primer Analysis software (Oligo7). Melting curve analysis was performed employing a 3-step analysis by: 1) selecting negative controls, 2) normalization of the melting curves, and 3) curve shift to normalize temperatures and group melt profiles of similar shape. PCR products displaying a melting curve that differs from the reference group were re-sequenced and were run on a 96-capillary Spectrumedix Sequencer. DNA samples from all 15 breast cancer patients were screened for genetic alterations in the entire coding sequence plus splice junction boundaries of BRCA1 and BRCA2. 51 and 31 known BRCA1 and BRCA2 heterozygous sequence variants were analyzed and compared with those of 10 healthy individuals. As a result, all types of single nucleotide variations, small deletions and insertions were easily detected by HRM analysis on the LightScanner-32 instrument. To determine the specificity of HRM analysis, a blind screening of 15 patients for BRCA1 and BRCA2 was performed. All 90 amplicons of 15 patients were screened by HRM analysis at the default sensitivity, and in parallel were sequenced. All sequence variants were detected on the LightScanner-32, confirming the 100% sensitivity and specificity of 98.96% because of 24 false positive samples confirmed by Sanger sequencing. Therefore, HRM analysis detected all known sequence variants, giving a sensitivity value of 100% and it could be undertaken in low- or middle-income countries as a high-throughput screening method of BRCA1 and BRCA2 and could be paired with proper genetic counseling to help affected women and their at-risk relatives understand the genetic risk factors of breast cancer. The results of the PP4 have been summarized in one article published in local journal.

Analysis of four pilot trials showed that the CMG scientists have made enormous progress during the planning, implementation, and R&D part of pilot trials which led to generation of engagement in strong and mutual research collaboration and training of Armenian scientists and project managers, strong research results, and clear ideas for the development of new proposals. Fortunately, the final discussion of results obtained in these pilot trials showed that the despite of small bottlenecks raised during their performance and accomplishment, CMG was able to provide high scientific and managerial potential for EC Horizon2020 proposal preparation and application.


Research Assessment of CMG
CMG was evaluated in accordance with the research assessment protocol developed by the Strategy Expert Group (SEG). That measured and reported on the effectiveness and impact of all programs developed, and on other activities undertaken by CMG, based on outcomes, including research, partnerships and public engagement, translation and use of knowledge, and organizational excellence. During the evaluation, a distinction was made between research, societal, and organizational perspectives. Quality in a research perspective was evaluated in terms of research results, publication, PhD production, R&D grants, and recruitment and training of researchers who can match the best in the field, and international research cooperation. Quality in a societal perspective was evaluated in terms of contributions to education, popular publishing and appearance in the media, participation in councils and boards. Quality in an organizational perspective was evaluated in terms of research management, training and professional qualification, human resource management, and access to equipment and other research resources. The research assessment was planned in accordance with an Action Plan that identified what were to be evaluated. Firstly, the SEG carried out a preliminary study to contribute to identifying specific strength, opportunities, challenges and risks. Then Terms of Reference were drawn up defining the frames of the evaluation. The Panel of Experts was appointed and involved in the SEG with responsibility for the assessment and recommendations which prepared a draft report. The SEG prepared the final report by the end of the project.

In the research assessment, it was recognized that CMG was charged with the responsibility of examining social, ethical and legal issues related to health, a responsibility that is particularly acute with the technical possibilities of modern medical genetics. Knowledge translation is also a major component of the mandate of CMG, a component that assures the society receives the benefit from its investment in health research. The current mandate of CMG in Armenia is to support research on the human genome and on all aspects of human genetics related to health and disease, including the translation of knowledge into health policy and practice, and the societal implications of genetic discoveries. Vision of CMG is to become the leading Armenian and regional institution for the advocacy, development and support of all fields of medical and human genetic research, maximizing the health opportunities offered by this knowledge to the benefit of Armenian community. The CMG’s mandate and structure are unique in the region. CMG supports research in medical and human genetics, health care services, and population health which are unique not only in Armenia but also in the whole region. The center is the only teaching body of medical genetics across the country. CMG is a part of a larger national research network that links researchers and other stakeholders across the country. CMG has a special responsibility and role to support research in fundamental biomedical science. The importance of genetics to all branches of health research therefore requires that CMG continuously facilitates and supports the genetic and genomic research. In these diverse roles, the activities of CMG are of significance to the entire health research community of Armenia. In conclusion, the research assessment has led to the identification of the following values of CMG: research excellence, investigator-initiated and innovative research, strong partnership and collaboration, public engagement and responsiveness, and recognition and leveraging of Armenian research strengths. Finally, the results of the research assessment have led to the recognition that CMG is a "bottom-up" organization. By responding to the needs of individual investigators and other stakeholders, the center ensured that its strategic goals and research priorities are consistent with the requirements of those who generate new knowledge and those who use it.

Development of the Strategy of the CMG
The action of SEG, including three SEG meetings and three visits to CMG, had provided guidance for the priority-setting process, and forged essential links with the research staff of the center. Through an iterative process of assessment and analysis, the SEG refined and informed the CMG’s Strategy which was updated by the assistance of the Europe for Business Ltd by reviewing the indicators of SWOT analysis and internal report. In its final version, the Strategy plan consisted of the following elements: Strategy in research in medical genetics by identifying four research priority areas of CMG; Strategy in grant funding and recruitment of scientists; Strategy in Education program in Medical Genetics; Strategy in Health Genetics Program; Strategy in Human Resources; Strategy in the CMG Infrastructure; and Strategy in the CMG Administration.

Recommendations and Actions Proposed
The SEG developed and released recommendations and actions addressed to the EC and Armenian authorities on Research and Development (R&D) cooperation of Armenia to increase its efficiency. The main recommendations for Armenia are based on the example of the CMG as an acting R&D center with recently known research assessment and developed strategy plan:


1. To effectively respond to the forces of globalization and achieve sustainable development, Armenia needs to build and/or strengthen institutions for Research and Development (R&D) which need to have clear indicators of acquired excellence status, including indicators for scientific outputs, output of services, for capability building, for delivery system/business development, for management, and general indicators of performance for the institute.

2. The establishment of Network of Centers of Excellence as one of the best ways and means of strengthening the country’s scientific and technological development. Essentially, it is meaningful for such developing country as Armenia is to build its individual scientific and technological capabilities through agencies and programs that consolidate and sharply focus their resources on common well-defined problems. Armenia has to establish or be actively involved in national and international networks of centers of excellence programs as partnerships among universities, industry, government and non-governmental organizations that focus on conducting and training scientific research and entrepreneurial capacity into tangible economic and social benefits for Armenia and its economic competitiveness. The program should mobilize and network distinguished scientists with peer reviewed scientific outputs to work together on common national research projects. A key goal of the initiative should be to mobilize and enable young Armenian scientists to engage in science and technological research.

3. Through research networking to other regional initiatives on centers of excellence, such centers, including CMG should draw lessons from European Union’s experiences in developing knowledge networks or centers of excellence. To achieve the above objectives, Armenian authorities in collaboration with competent regional and international institutions should:
• Undertake a study to provide conceptual understanding of what constitutes a center of excellence and the specific areas/fields of sustainable development in which such centers should be considered for strengthening and/or creation.
• Develop draft guidelines for identifying, developing, reviewing and evaluating large regional S&T facilities and networks of centers of excellence in Armenia.
• Set up supporting centers (such as genetic counseling centers for CMG) in the regions of Armenia to create awareness about the genetic conditions.
• Prepare a data bank and profiles of large science facilities in Armenia to identify existing science laboratories that offer opportunities for regional cooperation in specific areas.

4. Creation of population-wide biobanking. Chronic inflammatory background is acknowledged presently as a high risk factor for the major human non-communicable diseases (oncological, neurodegenerative and cardiovascular and diabetes). Based on the facts that Armenian population is affected by heavily penetrated genetic mutations associated with autoinflammatory diseases, there is a strategically important need in analysis of the overall impact of these genetic alterations in documented high levels of non-communicable diseases among Armenian population. Explorations in this area will largely contribute in developing preventive, early diagnostic and therapeutic interventions to combat the most deteriorating disorders in general and among Armenian population in a large extent. Establishment of a population-wide Biobank extended by IT-based database will be the first instrument in achieving this goal. The Biobanking will imply (i) Collection of biological samples from patients and healthy individuals along with available demographic and clinical records; (ii) Sample analysis (genomics, disease-specific biomarkers and routine cellular and biochemical tests); (iii) Establishment of a state of art IT facility for data storage and subsequent analysis and interpretation. Most likely, a high degree of preservation of the genetic background among Armenian population as a result of historical reasons may explain an extensive penetration of mutations in specific genes responsible for elevated inflammatory background in Armenians. For that reason the suggested Biobank/Database might be absolutely unparalleled anywhere in the world and will definitely have a significant impact on the Healthcare and medical research in Armenia.

5. Based on the large experience, valuable data accumulated during last decades as well as recent outcomes from the INARMERA pilot projects one may foresee a rational for CMG to extend its research and practical activity by enlarging a collaborative ties with research and medical units in Arab countries, Israel and Turkey as well as EU and USA where due to acceleration of migration events, penetration of the ethnic-based specific mutations is becoming more relevant. Formation/participation in multilateral Consortiums will be an excellent opportunity for promoting the Consortium’s a research on impact of genetically preconditioned inflammatory background on deteriorating human non-communicable diseases.

6. Health condition of a population actually influences not only wellbeing of an individual, but also, to a large extent, the future social and economic development and security of the nation and the country. Unfortunately, the figures reflecting the demographics of the world place Armenia among countries with very poor health condition and perspectives. Thus, Armenia is one of leading countries regarding the death rate from non-communicable diseases that could not be reasoned just by relatively low economic situation. In this regard, there will be a prerequisite for CMG to involve also the government of Armenia and venture capital in launching a strategically vital program to identify specific roots of high degree unhealthiness among Armenians.
These recommendations suggest that Armenian authorities should consider and use them in their national and regional efforts to develop centers of excellence making impact that is effectively and efficiently solving or contributing to the solution of specific national or regional problems.

Potential Impact:
The INARMERA project had an obvious potential impact for the scientific community as well as for the society. First of all the participation in the consortium provided the possibility of a professional growth for young scientists, PhD students and clinicians, who had the opportunity to travel to the laboratories of the involved European partners to gain specific expertise. The CMG as a research center recommended by the Government of Armenia has strongly profited from the project in several ways. The project that involved training and increasing the awareness of new medical genetics approaches in research and diagnostics, allowed the participation of young scientists and clinicians in courses that addressed different aspects of medical genetics. This knowledge was then further disseminated in Armenian scientific and clinical communities, and among the general public. The project participants could profit from newly established scientific and clinical contacts. The project also supported the participation of the partner teams in scientific conferences where they could present their findings, learn about the progress in the field, further discuss the methods and interpretation of the results, and find new collaborations. Young researchers and clinicians were preferentially supported to attend scientific and networking meetings.

In the scope of pilot trials, many Armenian professional collaborators were involved, further increasing the outreach of the project and the public awareness of addressed medical genetics problems. This preliminary part of the project brought a clear benefit also to several patients who were involved in the project and received a diagnosis explanation and course of further clinical management.

The INARMERA project helped to introduce Next Generation Sequencing analysis as the latest improvement in the genetic diagnostics for Armenian specialists. Thanks to the INARMERA project, there was an opportunity to introduce this new technology in Armenia, contributing to its demand and use. The use of such advanced molecular diagnostics tool in the definition of genetic disorders will eventually open a new phase in the prevention of the manifestations of many genetic disorders.

In order to raise awareness on medical genetics in general and make the CMG as a regional center of excellence in this field and on the project’s results specifically among the scientific community and the general public, the Consortium has developed a Networking and Dissemination plan based on the use of different channels. The INARMERA project website was set up by CMG (www.inarmeraproject.am). The main objective of the website was to increase public awareness about INARMERA. It was periodically updated with information regarding the events taking place in the CMG in the frame of INARMERA project. CMG has prepared INARMERA brochure as a key instrument for the Consortium promotion and awareness about the project. It features a brief overview of the project rationale, explaining the potential applications of INARMERA. An electronic copy of the brochure was also uploaded on the project website and is freely available. The CMG prepared a special brochure in Armenian entitled “Genetic tests in the health care system”, regarding the referrals for testing, professional genetic counseling, results of the genetic testing and indirect decisions. The brochure was mainly directed towards the general public and might serve as a guide for patients and their families, and was distributed to health care centers, professional organizations, and patient organizations around Armenia. For the dissemination of INARMERA results as well as the latest achievements of the CMG in regard with its research and genetic health priorities, the center has organized 4 special TV programs on the Public Television of Armenia between February-March 2013. A special flyer “Impact of spread of hereditary disorders in Europe as a result of migration from neighboring regions: Familial Mediterranean Fever” was developed and distributed at the Conference “Enhancement of European International Cooperation and Eastern Partnership in R&D&I“ organized under the Lithuanian Presidency of the Council of the European Union 30 September - 1 October, 2013 in Vilnius. Later it was also sent to the DG SANCO "Health Information" unit.

The Partners from the Consortium played an active role in the dissemination of the INARMERA Project information by carrying out a number of dissemination activities and by taking advance of their individual networks. Most of them attended different conferences and other events while disseminating the INARMERA Project through oral presentations and meetings. The European Society of Human Genetics Conferences in 2011, 2012, and 2013 were the central events to the dissemination strategy in Europe. The meetings facilitated contacts between people involved in all areas of human genetics coming from all over the world. During the conferences the Consortium had the opportunity to meet colleagues involved in the same fields of research and to share with them information and contents regarding the INARMERA activities and general policy. Open Information Day & Brokerage Event on FP7 Health in Brussels (9-10 June 2011) organized by the European Commission in the frames of the FP7 Health Partnering Event presented CMG as a successful institution during the plenary session by the event organizers and the INARMERA project information was highlighted in front of the all European health research community. Orphanet Europe Joint Action annual meetings in 2011, 2012, and 2013, where CMG hosted Armenia’s activities, contributed to the Joint Action, and presented the activities of Orphanet in Armenia. Existing European contacts were strengthened and new ones were established, particularly with INSERM. During the 6th European Conference on Rare Diseases & Orphan Products (24-25 May 2012, Brussels), Eurasian Conference on Rare Diseases & Orphan Products (21-23 June 2012, Moscow), and 26th Course in Medical Genetics (12-16 May 2013, Bologna) CMG specialists could explain main goals and achievements of the CMG in diagnostics and research of rare diseases through European collaboration thanks to INARMERA project, and build up new contacts for the future collaborations.

Activities through building cooperation and creating synergies with national and EU projects
In September 2011 the CMG set up cooperation with the Wilhelm Johannsen Centre, University of Copenhagen, Denmark in the field of rare and heterogeneous diseases associated with balanced chromosomal rearrangements (DBCR) and later in May 2013 - in the field of autoinflammatory disorders.
Through INARMERA project the Consortium could also participate in the European Molecular Genetics Quality Network (EMQN) workshop (Bruges, 18-19 September 2011) to obtain the final consensus document “Guidelines for the genetic diagnosis of Hereditary Periodic Fevers (HRF)” for genetic diagnostic testing of HRFs, reporting the genetic results and defining their clinical significance.
CMG participants gave three posters and one oral presentation at the Autoinflammation 2012 Congress organized by the International Society of Systemic Auto-Inflammatory Diseases (Lausanne, 22-26 May, 2013). CMG was also involved in the EuroFever project promoted by the Autoinflammatory Diseases’ Working Group of the Paediatric Rheumatology European Society. In the frame of this congress, the CMG set up new joint collaboration with the National Institute of Health of France (INSERM U 654).

Networking and Dissemination in Armenia and in the region
Immediately after the start of the INARMERA project (20 February 2011) CMG was invited to the FP7 National Information Point of Armenia at the National Academy of Sciences to present its two FP7 projects - CHERISH and INARMERA as a good practice cases to share with experience on participation in FP7 projects in front of the representatives of different Armenian research institutions applying for EU grants and in the presence of the European Commissars.
Rare Disease Day conferences of Armenia in 2012 and 2013 were an important auditoriums both for general public and health care professionals in Armenia to disseminate information about the current activities of CMG with special presentation and discussion about the INARMERA project.
CMG has set up and granted two new national research projects in collaboration with two different institutions - National Haematology Institute and “Arabkir” children hospital-research center. This allowed establishing new area of health care services for patients diagnosed with different forms of leukemia and autoinflammatory disorders in context of two research priority areas of CMG.
CMG also established a regional collaboration with Georgian Hematology and Blood Perfusion Scientific Research Institute (visited in June 2012). Two regional agreements for future collaboration had been created with Georgian Foundation for Genetic and Rare Diseases and Children Department at JSC Traumatology of Tbilisi for genetic and cytogenetic diagnostics of Georgian patients with rare diseases at CMG. CMG has established also collaboration with the Georgian National Forensic Laboratory (visited in November, 2013) for the improvement of scientific and methodical work, examination methods and implementation of new methods.

List of Websites:

www.inarmeraproject.am

P1) Center of Medical Genetics and Primary Health Care
Prof.Tamara Sarkisian – Coordinator
Tel: +37410544365
Fax:+37410544366
E-mail: tamsar@sci.am

P2) Danish Cancer Society
Prof. Eugene Lukanidin - Associated Research Leader
Tel: +4530382339
E-mail: el@cancer.dk

P3) European Centre for Knowledge and Technology Transfer
Dr. Ruben Vardapetian
Tel/Fax: +3222306916
E-mail: rv.eurotex@gmail.com