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Purpose: Approximately 1-2% of chronic myeloid leukemia (CML) patients harbor atypical BCR-ABL1 transcripts that cannot be monitored by real-time quantitative PCR (RT-qPCR) using standard methodologies. Within the European Treatment and Outcome Study (EUTOS) for CML we established and validated robust RT-qPCR methods for these patients., Methods: BCR-ABL1 transcripts were amplified and sequenced to characterize the underlying fusion. Residual disease monitoring was carried out by RT-qPCR with specific primers and probes using serial dilutions of appropriate BCR-ABL1 and GUSB plasmid DNA calibrators. Results were expressed as log reduction of the BCR-ABL1/GUSB ratio relative to the patient-specific baseline value and evaluated as an individual molecular response (IMR)., Results: In total, 330 blood samples (2-34 per patient, median 8) from 33 CML patients (19 male, median age 62 years) were analyzed. Patients expressed seven different atypical BCR-ABL1 transcripts (e1a2, n = 6; e6a2, n = 1; e8a2, n = 2; e13a3, n = 4; e14a3, n = 6; e13a3/e14a3, n = 2; e19a2, n = 12). Most patients (61%) responded well to TKI therapy and achieved an IMR of at least one log reduction 3 months after diagnosis. Four patients relapsed with a significant increase of BCR-ABL1/GUSB ratios., Conclusions: Characterization of atypical BCR-ABL1 transcripts is essential for adequate patient monitoring and to avoid false-negative results. The results cannot be expressed on the International Scale (IS) and thus the common molecular milestones and guidelines for treatment are difficult to apply. We, therefore, suggest reporting IMR levels in these cases as a time-dependent log reduction of BCR-ABL1 transcript levels compared to baseline prior to therapy., (© 2021. The Author(s).)

The purpose of this meta-study was to investigate β-thalassemia (β-thal) mutations and their chromosomal background in order to highlight the origin and spread of thalassemia alleles in the European and Mediterranean areas. Screening of more than 100 new Romanian β-thal alleles was also conducted. The results suggest an ancient introduction of mutations at codon 39 (C > T) (HBB: c.118C > T) and IVS-I-6 (T > C) (HBB: c.92 + 6T > C) in Romania. A comparative study was performed based on restriction fragment length polymorphism (RFLP) haplotypes associated with β-thal mutations in Romania and in Mediterranean countries. Each common β-thal allele from different populations exhibits a high degree of haplotype similarity, a sign of a clear unicentric origin for the IVS-I-110 (G > A) (HBB: c.93-21G > A), IVS-I-6, IVS-II-745 (C > G) (HBB: c.316-106C > G) and codon 39 mutations (the 17a [+ - - - - + +], 13c [ - + + - - - +], 17c [ + - - - - - +] and 14a [- + + - + + + ] ancestral RFLP background, respectively), followed by recurrent recombination events. This study also showed that geographic distances played a major role in shaping the spread of the predominant β-thal alleles, whereas no genetic boundaries were detected between broad groups of populations living in the Middle East, Europe and North Africa. The analyses revealed some discrepancies concerning Morocco and Serbia, which suggest some peculiar genetic flows. Marked variations in β(A) were observed between Southeast Asia and the Mediterranean, whereas a relative genetic homogeneity was found around the Mediterranean Basin. This homogeneity is undoubtedly the result of the high level of specific historic human migrations that occurred in this area.

Treatment of chronic myeloid leukemia (CML) with tyrosine kinase inhibitors has advanced to a stage where many patients achieve very low or undetectable levels of disease. Remarkably, some of these patients remain in sustained remission when treatment is withdrawn, suggesting that they may be at least operationally cured of their disease. Accurate definition of deep molecular responses (MRs) is therefore increasingly important for optimal patient management and comparison of independent data sets. We previously published proposals for broad standardized definitions of MR at different levels of sensitivity. Here we present detailed laboratory recommendations, developed as part of the European Treatment and Outcome Study for CML (EUTOS), to enable testing laboratories to score MR in a reproducible manner for CML patients expressing the most common BCR-ABL1 variants.

Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).

Background: Intrauterine growth restriction (IUGR) is a major cause of fetal morbidity and mortality during pregnancy. The role of mutation in the factor V gene, prothrombin gene, MTHFR gene, as risk factors for intrauterine growth restriction during pregnancy, is not very well known so far., Materials and Methods: This is a retrospective study of 151 pregnant women with a history of complicated pregnancy: intrauterine growth restriction, preeclampsia, recurrent pregnancy loss or maternal venous thromboembolism, who were admitted in Bucharest Emergency University Hospital, during the period January 2010 to July 2014. Genetic testing was performed for all the cases to detect: factor V Leiden mutation, G20210A mutation in the prothrombin gene, C677T mutation and A1298C mutation in methylenetetrahydrofolate reductase (MTHFR) gene. Blood samples were obtained as soon as the diagnosis of intrauterine growth restriction was established with ultrasonography., Results: The following gene mutations were associated with increased risk of IUGR: G20210A prothrombin gene mutation (OR 4.81, 95% CI 1.05 - 2.22, p= 0.043), G1691A factor V gene mutation (factor V Leiden) (OR 1.58, 95% CI 0.61 - 4.080, p= 0.347), C677T MTHFR gene mutation (OR 1.61, 95% CI 0.79 to 3.26, p= 0.186), compound heterozygous MTHFR C677T and A1298C (OR 1.66, 95% CI 0.81- 3.42, p= 0.169). Particularly, for G20210A prothrombin gene mutation we found statistically significant risk (p≤0.05) of IUGR.

Pregnancy is a normal physiological state that predisposes to thrombosis, determined by hormonal changes in the body. These changes occur in the blood flow (venous stasis), changes in the vascular wall (hypotonia, endothelial lesion) and changes in the coagulation factors (increased levels of factor VII, factor VIII, factor X, von Willebrand factor) and decreased activity levels of natural anticoagulants (protein C, protein S). In this study, we tried to determine a possible association between thrombosis and inherited thrombophilia in pregnant women. This is a retrospective study of 151 pregnant women with a history of complicated pregnancy: maternal thrombosis and placental vascular pathology (intrauterine growth restriction, preeclampsia, recurrent pregnancy loss), who were admitted in our hospital during the period January 2010 to July 2014. We performed genetic analyses to detect the factor V Leiden mutation, the G20210A mutation in the prothrombin gene, the C677T mutation and the A1298C mutation in methylenetetrahydrofolate reductase (MTHFR) gene. The risk of thrombosis in patients with factor V Leiden is 2.66 times higher than the patients negative for this mutation (OR 2.66 95% CI 0.96-7.37 P=0.059). We did not find any statistical association with mutations in the MTHFR gene. Pregnant women with a family history of thrombosis present a 2.18-fold higher risk of thrombosis (OR 2.18 CI 0.9-5.26 P=0.085). Of 151 pregnant women, thrombotic events occurred in 24 patients: deep vein thrombosis, pulmonary embolism, cerebral venous sinus thrombosis and ischemic stroke. The occurrence of thrombotic events was identified in the last trimester of pregnancy, but especially postpartum. Thrombosis in pregnancy is a redoubtable complication requiring an excellent cooperation between the obstetrician and anesthesiologist.

Thalassaemia major is a classical example of a disease that can be prevented by prenatal diagnosis. In Romania there are currently 300 patients with thalassaemia major under the management of specialized institutions. Prenatal diagnoses of thalassemia have offered a new dimension to the prevention of this disease, but in order to implement prenatal diagnosis, knowledge of mutations and of their incidence is essential. Molecular testing using Denaturing Gradient Gel Electrophoresis (DGGE) scanning and direct mutation detection with Amplificaton Refractory Mutation System-PCR (ARMS-PCR) and Restriction endonuclease Analysis of PCR fragments (PCR-RFLP) was performed by using amplified DNA from amniotic cells samples, while mutations in the parents were determined in advance. Using our experience in molecular diagnosis, we were able to perform the first prenatal diagnosis for two young couples at risk for thalassaemia major. Foetal samplings were collected by amniocentesis and chorionic villus sampling in the second trimester of the pregnancies. Maternal contamination of the foetal DNA was ruled out by STR genotyping. The prenatal diagnosis revealed affected foetuses with homozygous status of beta-thalassemia major. The IVSI-110 (G-A)/IVS II-745 (C-G) genotype in the first case foetus and ed 8 (-AA)/cd 8 (-AA) in the second case foetus were reported. The results of this study point to a successful future prenatal diagnosis of beta-thalassnemia in Romania, using a rapid and accurate molecular method. Together with the implementation of proper preventive health measures and the education of parents regarding their carrier status, we are hoping that this method will be used as the common application approach to decrease the incidence of thalassacmia major.

Beta-thalassemia is uncommon (0.5%) in the Romanian population, but it must be considered in the differential diagnosis of hypochromic anemia. The molecular characterization of beta-thalassemia is absolutely necessary for molecular diagnosis, as well as any genetic epidemiological study in this region. Molecular analyses consist of mutation detection by molecular scanning of beta-globin gene. This gene has 3 exons and 2 introns, involved in beta-thalassemic pathogenesis. Clinical application of DNA analysis on beta-thalassemic chromosomes allowed characterization of 29 persons with different beta-thalassemia mutations among 58 patients with anemia. The experimental strategy was based on sequential PCR amplification of most of the beta-globin gene and running on denaturing gradient gel electrophoresis of amplification products. Definitive characterization of mutations in samples identified with shifted DGGE patterns was performed ARMS-PCR and/or PCR-restriction enzyme analysis methods. Eight different beta-thalassemia alleles were identified, the most common being IVS I-110 (G-A) and cd 39 (C-T). Comparison of overall frequency of mutations in the neighboring countries, shows that these results are in the frame of overall distribution of these mutations in Mediterranean area, especially in Greece and in Bulgaria. Molecular diagnosis is useful for differentiating mild from severe alleles, for genetic counseling, as well as for mutation definition in carriers, identified by hematological analysis necessary for prenatal testing and genetic counseling.