A Highly Sensitive Quantitative Real-Time PCR Assay for Determination of Mutant JAK2 Exon 12 Allele Burden

Mutations in the Janus kinase 2 (JAK2) gene have become an important identifier for the Philadelphia-chromosome negative chronic myeloproliferative neoplasms. In contrast to the JAK2V617F mutation, the large number of JAK2 exon 12 mutations has challenged the development of quantitative assays. We present a highly sensitive real-time quantitative PCR assay for determination of the mutant allele burden of JAK2 exon 12 mutations. In combination with high resolution melting analysis and sequencing the assay identified six patients carrying previously described JAK2 exon 12 mutations and one novel mutation. Two patients were homozygous with a high mutant allele burden, whereas one of the heterozygous patients had a very low mutant allele burden. The allele burden in the peripheral blood resembled that of the bone marrow, except for the patient with low allele burden. Myeloid and lymphoid cell populations were isolated by cell sorting and quantitative PCR revealed similar mutant allele burdens in CD16+ granulocytes and peripheral blood. The mutations were also detected in B-lymphocytes in half of the patients at a low allele burden. In conclusion, our highly sensitive assay provides an important tool for quantitative monitoring of the mutant allele burden and accordingly also for determining the impact of treatment with interferon-α-2, shown to induce molecular remission in JAK2V617F-positive patients, which may be a future treatment option for JAK2 exon 12-positive patients as well

Novel mutations and their functional and clinical relevance in myeloproliferative neoplasms: JAK2, MPL, TET2, ASXL1, CBL, IDH and IKZF1

Myeloproliferative neoplasms (MPNs) originate from genetically transformed hematopoietic stem cells that retain the capacity for multilineage differentiation and effective myelopoiesis. Beginning in early 2005, a number of novel mutations involving Janus kinase 2 (JAK2), Myeloproliferative Leukemia Virus (MPL), TET oncogene family member 2 (TET2), Additional Sex Combs-Like 1 (ASXL1), Casitas B-lineage lymphoma proto-oncogene (CBL), Isocitrate dehydrogenase (IDH) and IKAROS family zinc finger 1 (IKZF1) have been described in BCR-ABL1-negative MPNs. However, none of these mutations were MPN specific, displayed mutual exclusivity or could be traced back to a common ancestral clone. JAK2 and MPL mutations appear to exert a phenotype-modifying effect and are distinctly associated with polycythemia vera, essential thrombocythemia and primary myelofibrosis; the corresponding mutational frequencies are ∼99, 55 and 65% for JAK2 and 0, 3 and 10% for MPL mutations. The incidence of TET2, ASXL1, CBL, IDH or IKZF1 mutations in these disorders ranges from 0 to 17% these latter mutations are more common in chronic (TET2, ASXL1, CBL) or juvenile (CBL) myelomonocytic leukemias, mastocytosis (TET2), myelodysplastic syndromes (TET2, ASXL1) and secondary acute myeloid leukemia, including blast-phase MPN (IDH, ASXL1, IKZF1). The functional consequences of MPN-associated mutations include unregulated JAK-STAT (Janus kinase/signal transducer and activator of transcription) signaling, epigenetic modulation of transcription and abnormal accumulation of oncoproteins. However, it is not clear as to whether and how these abnormalities contribute to disease initiation, clonal evolution or blastic transformation

Investigating the roots of political disengagement of young Greek Cypriots

This study attempted to disentangle the issues underlying the marked drop recorded in political engagement of young Greek Cypriots. To reveal the dynamic processes through which people debate, disagree or convince each other towards the formation of political attitudes, eight focus-groups were carried out with a total of forty participants, equally distributed according to age and gender. The analysis showed that young Greek Cypriots appear uncertain for their future, pessimistic, cynical, and highly disillusioned with traditional politics. Contrary to what is observed in other European countries, young Greek Cypriots do not experiment with alternative forms of political action, remain inactive, and although the country's politicised culture of the past is still reflected in their theoretical discussions about social issues, they express embarrassment and confusion when asked to elaborate on how theory could be transformed into practice. They associate politics with corruption and economic interests, they are scornfully disillusioned with the European Union, and they emotionally distance themselves from important changes to come with the possible reunification of Cyprus. Young Greek Cypriots appear insecure, pessimistic, disoriented, uninspired, and in an urgent need to rediscover passion for ideas which they cannot any more find in traditional politics and forms of political action. © 2017 Academy of Social Sciences

Delivery of oligonucleotides to bone marrow to modulate ferrochelatase splicing in a mouse model of erythropoietic protoporphyria

Erythropoietic protoporphyria (EPP) is a rare genetic disease in which patients experience acute phototoxic reactions after sunlight exposure. It is caused by a deficiency in ferrochelatase (FECH) in the heme biosynthesis pathway. Most patients exhibit a loss-of-function mutation in trans to an allele bearing a SNP that favors aberrant splicing of transcripts. One viable strategy for EPP is to deploy splice-switching oligonucleotides (SSOs) to increase FECH synthesis, whereby an increase of a few percent would provide therapeutic benefit. However, successful application of SSOs in bone marrow cells is not described. Here, we show that SSOs comprising methoxyethyl-chemistry increase FECH levels in cells. We conjugated one SSO to three prototypical targeting groups and administered them to a mouse model of EPP in order to study their biodistribution, their metabolic stability and their FECH splice-switching ability. The SSOs exhibited distinct distribution profiles, with increased accumulation in liver, kidney, bone marrow and lung. However, they also underwent substantial metabolism, mainly at their linker groups. An SSO bearing a cholesteryl group increased levels of correctly spliced FECH transcript by 80% in the bone marrow. The results provide a promising approach to treat EPP and other disorders originating from splicing dysregulation in the bone marrow.ISSN:1362-4962ISSN:0301-561

D-NA4.1 Functional Scenarios:WP5 Deliverable D5.1: D-NA4.1 Functional Scenarios

This deliverable describes the work conducted in ERIGrid 2.0 task NA4.1 ’Definition of Functional Scenarios’. The work has been conducted via a survey and a brainstorming workshop. The results are six Functional Scenarios: Ancillary services provided by Distributed Energy Resources (DERs) and active grid assets, Microgrids & energy communities, Sector coupling, Frequency and voltage stability in inverter dominated power systems, Aggregation and flexibility management, and Digitalisation, which describe the overarching topics within ERIGrid 2.0. The Functional Scenarios will be used as an input in further ERIGrid 2.0 work. Smart grid and smart energy systems solutions have become complex and multidisciplinary. With the further integration of Information and Communication Technology (ICT) and other energy systems new testing scenarios, profiles, and processes must be defined. In order to achieve this, big trends affecting research, testing, and validation processes have been reviewed, with a special focus on new aspects such as interoperability testing or digitalisation. The scenario descriptions define requirements, actors, etc. on a functional level. ERIGrid 2.0 work package NA4 ’Iterative Creation of Scenarios and Test Case Profiles’ addresses these needs. This work has been conducted with emphasis on the alignment with the European Green Deal, further support on the technology validation and roll-out phases, and further integration of the research infrastructures. A Functional Scenario has been defined as an umbrella term comprising of motivation and relevance for ERIGrid 2.0, system descriptions, use case and test case descriptions, and experimental setup descriptions. Each scenario has a single core idea and is formed on the basis of inclusiveness. Functional Scenarios consider several high-level scenarios in other projects and networks as a background forming the overall circumstances in which the Functional Scenario is considered. The high-level scenarios provide a holistic understanding of the current status and development while also highlighting future visions and requirements impacting the Functional Scenarios. The high-level scenarios also address the high-level drivers for the Functional Scenarios, such as needs for digitalisation of the smart energy systems. Furthermore, Functional Scenarios are related to the generic system configurations developed in ERIGrid and consider the work conducted in ERIGrid as a strong background for ERIGrid 2.0. The necessity for a mutual understanding of scenarios which are of interest to the ERIGrid 2.0 partners and their research infrastructures and in alignment of the project objectives, led to conducting a survey regarding the first actions of the NA4.1 work. The purpose of this survey was to gather inputs on a set of Functional Scenarios that were analysed in more detail to deduce the most relevant approaches for ERIGrid 2.0. Overall, 15 partners participated in the survey and submitted 35 scenarios. The survey results include scenarios on sector coupling, multi-energy systems, ICT and automation, energy communities, microgrids and low- inertia grids, and stability, control and grid code challenges. Detailed descriptions of Functional Scenarios submitted to the survey are presented in Appendix A: Functional Scenario Survey Data of this deliverable. The formation of the Functional Scenarios was organised in six working groups, each of which focused on a single Functional Scenario. The decision on the six Functional Scenario was taken during the NA4 regular meetings and the brainstorming workshop itself based on the results of the Functional Scenario survey. The focus of the first working group has been on a component focused scenario developed based on the survey results on DERs and inverters. The resulting Functional Scenario 1 integrates key components, such as DER inverters and controllers with ICT, control and automation architectures to enable new grid services with the development of interfaces between the active components. The second working group has been focused on topics related to microgrids and energy communities forming Functional Scenario 2 to support the local microgrid and energy community development by enabling flexibility services locally with ICT and control including exploitation of grid intelligence. While the third working group has been working on the survey results on sector coupling and multi-energy systems with Functional Scenario 3 anticipating a massive roll-out of power-to-X components in the near future by developing system level understanding of the impacts on the electrical domain. The fourth working group has been focused on grid management and overall the perspectives of Distribution System Operators (DSOs) and Transmission System Operators (TSOs) resulting in Functional Scenario 4 assuring frequency and voltage stability in low inertia systems through capabilities of Renewable Energy Sources (RES), Distributed Generation (DG), controllable loads and storage systems as well as ICT and control systems. The fifth working group has been based on the survey results comprising of aggregation, flexibility, market and reserve topics and defined Functional Scenario 5 to focus on communication functionality for aggregation, service matching, fail-over, configuration, and interoperability addressing scale-related properties of aggregation and control solutions. Lastly, the sixth working group has been focused on digitalisation including wide range of topics such as ICT infrastructure, communication, automation, control and monitoring. Functional Scenario 6 explores the impact of ICT solutions on the physical (electrical power) system covering new applications of data and data processing as well as new paths for exchanging data. The Functional Scenario templates used during the brainstorming workshop have been included in the Appendix B: Functional Scenario Templates. The work started in NA4.1 will continue in NA4.2 and NA4.4 with discussions on more detailed definitions of the test cases which will initially provide the inputs for other project activities. The discourse on the Functional Scenarios is also assumed to support ERIGrid 2.0 physical lab and virtual access work and decision-making beyond ERIGrid 2.0