Reply to Ghirardello et al Letter to the Editor

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DETECTION OF CALR MUTATIONS USING HIGH RESOLUTION MELTING CURVE ANALYSIS (HRM-A); APPLICATION ON A LARGE COHORT OF GREEK ET AND MF PATIENTS

Background and Objectives Somatic mutations in the calreticulin gene (CALR) are detected in approximately 70% of patients with essential thrombocythemia (ET) and primary or secondary myelofibosis (MF), lacking the JAK2and MPLmutations. To determine the prevalence of CALRframeshift mutations in a population of MPN patients of Greek origin, we developed a rapid low-budget PCR-based assay and screened samples from 5 tertiary Haematology units. This is a first of its kind report of the Greek patient population that also disclosed novel CALRmutants.   Methods MPN patient samples were collected from different clinical units and screened for JAK2and MPLmutations after informed consent was obtained. Negative samples were analyzed for the presence of CALRmutations. To this end, we developed a modified post Real Time PCR High Resolution Melting Curve analysis (HRM-A) protocol. Samples were subsequently confirmed by Sanger sequencing.   Results Using this protocol we screened 173 MPN, JAK2and MPLmutation negative, patients of Greek origin, of whom 117 (67.63%) displayed a CALRexon 9 mutation. More specifically, mutations were detected in 90 out of 130 (69.23%) essential thrombocythaemia cases (ET), in 18 out of 33 (54.55%) primary myelofibrosis patients (pMF) and in 9 out of 10 (90%) cases of myelofibrosis secondary to ET (post-ET sMF). False positive results were not detected. The limit of detection (LoD) of our protocol was 2%. Furthermore, our study reavealed 6 rare novel mutations which are to be added in the COSMIC database.    Conclusions Overall, our method could rapidly and cost-effectively detect the mutation status in a representative cohort of Greek patients; the mutation make-up in our group was not different from what has been published for other national groups

Genetic prediction of ICU hospitalization and mortality in COVID-19 patients using artificial neural networks

There is an unmet need of models for early prediction of morbidity and mortality of Coronavirus disease-19 (COVID-19). We aimed to a) identify complement-related genetic variants associated with the clinical outcomes of ICU hospitalization and death, b) develop an artificial neural network (ANN) predicting these outcomes and c) validate whether complement-related variants are associated with an impaired complement phenotype. We prospectively recruited consecutive adult patients of Caucasian origin, hospitalized due to COVID-19. Through targeted next-generation sequencing, we identified variants in complement factor H/CFH, CFB, CFH-related, CFD, CD55, C3, C5, CFI, CD46, thrombomodulin/THBD, and A Disintegrin and Metalloproteinase with Thrombospondin motifs (ADAMTS13). Among 381 variants in 133 patients, we identified 5 critical variants associated with severe COVID-19: rs2547438 (C3), rs2250656 (C3), rs1042580 (THBD), rs800292 (CFH) and rs414628 (CFHR1). Using age, gender and presence or absence of each variant, we developed an ANN predicting morbidity and mortality in 89.47% of the examined population. Furthermore, THBD and C3a levels were significantly increased in severe COVID-19 patients and those harbouring relevant variants. Thus, we reveal for the first time an ANN accurately predicting ICU hospitalization and death in COVID-19 patients, based on genetic variants in complement genes, age and gender. Importantly, we confirm that genetic dysregulation is associated with impaired complement phenotype.- Pfizer Pharmaceuticals(undefined

Study of pharmacological agents that induce γ - chain grobin expression in human erythroid cultures

Current treatment of thalassemia and sickle cell anemia includes inducers of hemoglobin F synthesis. However, because of concerns about the dose limiting myelotoxicity, potential carcinogenicity and high cost of the agents, an intensive search for less toxic or more effective drugs is ongoing. In this study we tested the effect of valproic acid and trichostatin, alone or in combination with hemin, on γ chain synthesis in human erythroid liquid cultures. The agents were tested on erythroid human liquid cultures derived from normal peripheral blood, peripheral blood from β⁵/βᵗʰᵃˡ patients, normal cord blood and normal bone marrow samples. The effect of the agents was expressed as increase of γ/γ+β m-RNA, measured with competitive reverse transcriptase-polymerase chain reaction (RT-PCR), or as increase of HbF, measured by high performance liquid chromatography (HPLC). Addition of valproic acid or trichostatin to human erythroid cell cultures enhanced preferentially enhanced γ mRNA synthesis in all blood samples (2.9-3,5 fold). The addition of hemin enhanced the effect up to 10 fold. Valproic acid, trichostatin and their combination with hemin (all three FDA approved drugs) preferentially increase γ globin chain synthesis and may be helpful for the treatment of hemoglobinopathies.Τα τελευταία χρόνια, για τη θεραπεία των θαλασσαιμικών συνδρόμων (β-ΜΑ, δρεπανοκυτταρική αναιμία) εκτός από τη συμβατική θεραπεία χρησιμοποιούνται και φαρμακευτικές ουσίες που προκαλούν επαγωγή της HbF. Στη συγκεκριμένη μελέτη ελέγξαμε τη δράση του βαλπροϊκού, της τριχοστατίνης και της αιμίνης στην έκφραση των γ-αλύσων της αιμοσφαιρίνης σε ανθρώπινες ερυθροποιητικές καλλιέργειες που προέρχονται από δείγματα περιφερικού αίματος φυσιολογικών ατόμων και μικροδρεπανοκυτταρικών ασθενών, δείγματα φυσιολογικού ομφάλιου λώρου και μυελού των οστών. Η δράση των ουσιών εκφράστηκε ως αύξηση του λόγου γ/γ+β mRNA (που προσδιορίστηκε με την ανταγωνιστική ανάστροφη αλυσιδωτή αντίδραση πολυμεράσης – RI-PCR), ή ως αύξηση της HbF όπως αυτή μετράται με υγρή χρωματογραφία υψηλής απόδοσης (HPLC). Η προσθήκη του βαλπροϊκού ή της τριχοστατίνης εκλεκτικά επάγει την έκφραση των γ-αλύσεων (γ-mRNA) στα δείγματα από όλες τις πηγές (2.9-3,5 φορές). Η προσθήκη της αιμίνης προκάλεσε επαγωγή μέχρι 10 φορές. Το βαλπροϊκό, η τριχοστατίνη και ο συνδυασμός τους με την αιμίνη, εκλεκτικά επάγει την έκφραση των γ-αλύσεων και μπορεί να είναι χρήσιμος για τη θεραπεία των θαλασσαιμιών συνδρόμων

Genetic Polymorphisms Implicated in Major Pregnancy Complications: a Review

Pregnancy short- or long-term complications may involve the mother’s health, the fetus’s health, or both. A systematic literature review was performed, including studies up to October 2018 from Medline (PubMed), Science Direct, Web of Science and Google Scholar. The following inclusion criteria were applied: studies published until 2018 concerning the genetic background of pregnancy complications such as high blood pressure, gestational diabetes, preeclampsia, pregnancy loss, endometrial death, placental abruption, premature labor, and intrauterine growth retardation which may render pregnancy a high risk condition.We identified 164 articles that met the inclusion criteria and reviewed and analyzed them. The results so far are contradictory and the pathogenicity of these pregnancy complications remains unclear. For most of the polymorphisms studied so far, data refer to small studies size but research is on-going.The identification of genetic polymorphisms with strong correlations with certain pregnancy complications could provide us with useful tools which could be incorporated in diagnostic algorithms that could facilitate early detection and treatment of major pregnancy complications

Current trends in platelet transfusions practice: The role of ABO-RhD and human leukocyte antigen incompatibility

Platelet transfusions have contributed to the revolutionary modern treatment of hypoproliferative thrombocytopenia. Despite the long-term application of platelet transfusion in therapeutics, all aspects of their optimal use (i.e., in cases of ABO and/or Rh (D incompatibility) have not been definitively determined yet. We reviewed the available data on transfusion practices and outcome in ABO and RhD incompatibility and platelet refractoriness due to anti-human leukocyte antigen (HLA) antibodies. Transfusion of platelets with major ABO-incompatibility is related to reduced posttransfusion platelet (PLT) count increments, compared to ABO-identical and minor, but still are equally effective in preventing clinical bleeding. ABO-minor incompatible transfusions pose the risk of an acute hemolytic reaction of the recipient that is not always related to high anti-A, B donor titers. ABO-identical PLT transfusion seems to be the most effective and safest therapeutic strategy. Exclusive ABO-identical platelet transfusion policy could be feasible, but alternative approaches could facilitate platelet inventory management. Transfusion of platelets from RhD positive donors to RhD negative patients is considered to be effective and safe though is associated with low rate of anti-D alloimmunization due to contaminating red blood cells. The prevention of D alloimmunization is recommended only for women of childbearing age. HLA alloimmunization is a major cause of platelet refractoriness. Managing patients with refractoriness with cross-matched or HLA-matched platelets is the current practice although data are still lacking for the efficacy of this practice in terms of clinical outcome. Leukoreduction contributes to the reduction of both HLA and anti-D alloimmunization

Central Retinal Vein Occlusion Secondary to Clomiphene Treatment in a Male Carrier of Factor V Leiden

We report a case of a 35-year-old previously healthy man treated with clomiphene for infertility, who presented with blurred vision in his left eye due to ocular vein occlusion as documented by fluorescein angiography. The patient was heterozygous for the factor V Leiden (FV Leiden) mutation and for the 1298 A-C polymorphism of the methylene-tetrahydrofolate reductase (MTHFR) gene. He was treated with clopidogrel and is now free of symptoms. Because congenital thrombophilia is a moderate risk factor for central retinal vein occlusion and the administration of clomiphene may trigger this process, we recommend screening of young patients for FV Leiden before clomiphene treatment

Enhancing the Accuracy of Platelet to Lymphocyte Ratio after Adjustment for Large Platelet Count: A Pilot Study in Breast Cancer Patients

Background. The objective of our study is to investigate the potential effect of adjusting preoperative platelet to lymphocyte ratio, an emerging biomarker of survival in cancer patients, for the fraction of large platelets. Methods. A total of 79 patients with breast neoplasias, 44 with fibroadenomas, and 35 with invasive ductal carcinoma were included in the study. Both conventional platelet to lymphocyte ratio (PLR) and the adjusted marker, large platelet to lymphocyte ratio (LPLR), were correlated with laboratory and histopathological parameters of the study sample. Results. LPLR elevation was significantly correlated with the presence of malignancy, advanced tumor stage, metastatic spread in the axillary nodes and HER2/neu overexpression, while PLR was only correlated with the number of infiltrated lymph nodes. Conclusions. This is the first study evaluating the effect of adjustment for large platelet count on improving PLR accuracy, when correlated with the basic independent markers of survival in a sample of breast cancer patients. Further studies are needed in order to assess the possibility of applying our adjustment as standard in terms of predicting survival rates in cancer

A Successful Mother and Neonate Outcome for a Woman with Essential Thrombocytosis and FV Leiden Heterozygosity

Essential thrombocytosis (ET) and FV Leiden heterozygosity represent an acquired and hereditable hypercoagulable state, respectively. An uncommon case of coexistence of ET and FV Leiden heterozygosity in a 36-year-old pregnant woman and her successful pregnancy outcome is described. She was considered to be at high risk of thrombosis during her pregnancy and she was treated with both prophylactic dose of LMWH and aspirin daily throughout her pregnancy and for a 6-week period postpartum. The efficacy of the anticoagulation treatment was monitored in various time points not only by measuring anti-Xa levels and D-Dimers but also with new coagulation methods such as rotation thromboelastometry and multiplate. Global assessment of coagulation using additional newer laboratory tests might prove useful in monitoring coagulation pregnancies at high risk for thrombosis