Association of CYP3A5*3, CYP3A4*18 & CYP2B6*6 polymorphisms with imatinib treatment outcome in Azerbaijani chronic myeloid leukaemia patients.

Background & objectives: Imatinib mesylate (IM) is a reliable first line treatment for chronic myeloid leukaemia (CML). Nevertheless, despite promising results, a considerable proportion of patients develop resistance to the drug. Cytochrome P450 (CYP) enzymes play a crucial role in IM metabolism. Thus, point mutations in CYP genes may modify IM enzyme activity resulting in insufficient treatment response. This investigation was aimed to identify the functional impact of CYP3A5*3, CYP3A4*18 and CYP2B6*6 polymorphisms on the IM response in patients with CML in Azerbaijan. Methods: Genotyping of CYP3A5*3, CYP3A4*18 and CYP2B6*6 was performed in 153 patients (102 IM non-responders and 51 IM responders) with CML by the PCR-restriction fragment length polymorphism (RFLP) assays. The odds ratios (ORs) with 95 per cent confidence intervals (CIs) were applied to assess the association between allelic variants and IM therapy outcome. The results were validated by sequencing. Results: The frequency of the CYP3A4*18 allele was considerably lower in the responder's group (97.1 vs. 100%; P=0.036). For CYP3A5*3, the allelic frequency was slightly higher among the IM responders (100 vs. 99.02%) with no significant difference. Although patients heterozygous (TC) for CYP2B6*6 demonstrated a higher risk of acquiring resistance (OR 1.04; 95% CI: 0.492-2.218), differences were not significant (P=0.909). In addition, the homozygous genotype (TT) demonstrated a lower risk of unresponsiveness (OR 0.72; 95% CI: 0.283-1.836), but associations were not significant (P=0.491). Interpretation & conclusions: Our results demonstrated that CYP3A4*18 was significantly associated with IM treatment response in patients with CML in Azerbaijan, whereas rather common CYP3A5*3 was identified to have no such association.

Spectrum of BCR-ABL mutations in Azerbaijanian imatinib-resistant patients with chronic myeloid leukemia.

Objective: BCR-ABL1 kinase domain (KD) mutations can lead to resistance to first- and second-generation tyrosine kinase inhibitors (TKIs) in chronic myeloid leukemia (CML). Here, we present the first report of the spectrum of mutations in the BCR-ABL1 KD of CML patients from Azerbaijan. Materials and methods: Samples for mutation screening were obtained from patients experiencing resistance to first line TKIs or from patients in acceleration phase (AP) or blast crisis (BC) at the time of diagnosis. The cDNA region corresponding to BCR-ABL1 KD was sequenced by pyrosequencing method. The χ2 test was used to assess the association of categorical variables between mutation-positive and -negative groups. In addition, the Kaplan-Meier method was applied to generate survival curves. Results: Eight different point mutations were identified in 22 (13.4%) out of 163 CML patients experiencing resistance to TKIs. The types of mutations detected were as follows: Contact binding site mutations 50% (11), SH2 domain mutations 27.4% (six), P-loop mutations 18.1% (four), and SH3 domain mutations accounting for 4.5% (one). The most common mutation was T315I, accounting for 5% (n = 8) of all patients. Significant association was identified between BCR-ABL1 mutations and additional chromosomal aberrations as well as between the mutations and disease phases (p < 0.05). Twelve out of 22 patients with BCR-ABL1 mutations and seven out of eight with T315I were in BC. Overall survival (OS) of the patients with BCR-ABL1 mutations was significantly lower comparing to the patients with no mutation (p < 0.05) and 8 patients with T315I mutation presented OS of 0%. Conclusion: T315I was the most commonly identified BCR-ABL1 mutation in TKI-resistant CML patients of Azerbaijani origin, being associated with disease progression and poor OS.

First Report of ß-Thalassemia Intermedia in a Patient Compound Heterozygous for -92 (C>T) and Codons 36/37 (-T) Mutations.

The -92 (C>T) (HBB: c.-142C>T) is a silent ß-thalassemia (ß-thal) mutation previously described in combination with several ß0 mutations and expressed as ß-thal intermedia (ß-TI). Heterozygous individuals are known to be completely asymptomatic showing borderline hemoglobin (Hb), mean corpuscular volume (MCV) and Hb A2 levels. Here, we report the first incidence of -92 in Eastern Europe and in Azerbaijan, and the first case in combination with codons 36/37 (-T) (HBB: c.112delT) mutation.

Molecular and geographical heterogeneity of hemoglobinopathy mutations in Azerbaijanian populations.

With the carrier rate of 4%-8.6%, ß-thalassemia is one of the most prevalent hereditary disorders in Azerbaijan. Taking into consideration the high frequency of ß-thalassemia as well as the occurrences of several other hemoglobinopathies, we conducted a large genotyping study to investigate the mutational background of common hemoglobinopathies in the country. Α- and ß-globin genes were evaluated in the carriers of mutations identified via hematological indices and hemoglobin fractions (n = 1,757). Genotyping of ß-thalassemia carriers identified through population screening revealed 32 mutations, with codon 8 [-AA]-34.96%, IVS-II-1 [G > A]-16.35%, and IVS-I-110 [G > A]-10.12% leading the spectrum. Analysis of associations of ß-thalassemia mutations with geographical regions of the country identified the strongest association between codon 8 [-AA] and Shaki-Zaqatala, and codon 5 [-CT] in Mountainous Shirvan regions (ri > 6.00; p < 0.05). HbS, HbD-Punjab, and HbE were the most prevalent among our variant hemoglobin cohort, commonly inherited in compounds with ß-thalassemia than in the homozygous state. We identified nine α-thalassemia mutations, 20.5 kb and 3.7 kb deletions together accounting for 74% of the spectrum. Point mutations of α-thalassemia were less common among our observations and were mainly inherited in compounds with deletions. Our results allow a better understanding of the wide spectrum of mutations in Azerbaijan and highlights the high heterogeneity of hemoglobinopathies in the local population.

A Novel Frameshift Mutation at Codon 2 (-T) (HBB: c.9delT) and First Report of Three New ß-Globin Mutations From Azerbaijan.

We identified a novel mutation of ß-thalassemia (ß-thal) in a heterozygous carrier from Azerbaijan. Phenotypical data and molecular mechanisms of codon 2 (-T) (HBB: c.9delT) was relevant to ß0-thal. Additionally, we here report two new mutations on the HBB gene, not observed previously, in the local population as well as a non causative promoter mutation -198 (A>G) (HBB: c.-248A>G).

Codon 14 (+T) (HBB: c.44_45insT): a Rare ß-Thalassemia Mutation Reported Only in Azerbaijan.

Codon 14 (+T) (HBB: c.44_45insT) is a very rare ß-thalassemia (ß-thal) mutation previously reported in three ß-thal major (ß-TM) patients of Azerbaijani origin. None of the previous reports described the genotype-phenotype correlation of the mutation. We here report the first case of homozygous codon 14 together with data of the heterozygous parents.

Thalassemia in the laboratory: pearls, pitfalls, and promises.

Thalassemia is one of the most common hereditary disorders of the developing world, and it is associated with severe anemia and transfusion dependence. The global health burden of thalassemia has increased as a result of human mobility and migration in recent years. Depending on inherited mutations, thalassemia patients exhibit distorted hemoglobin (Hb) patterns and deviated red cell indices, both of which can be used to support identification by diagnostic tools. Diagnostic approaches vary depending on the target population and the aim of the testing. Current methods, which are based on Hb patterns, are used for first-line screening, whereas molecular testing is needed for conformation of the results and for prenatal and preimplantation genetic diagnosis. In the present paper, we review the diagnostic parameters, pitfalls, interfering factors, and methods; currently available best-practice guidelines; quality assurance and standardization of the procedures; and promising laboratory technologies for the future of thalassemia diagnosis.

ß-Thalassemia intermedia: a comprehensive overview and novel approaches.

ß-Thalassemia intermedia is a clinical condition of intermediate gravity between ß-thalassemia minor, the asymptomatic carrier, and ß-thalassemia major, the transfusion-dependent severe anemia. It is characterized by a significant clinical polymorphism, which is attributable to its genetic heterogeneity. Ineffective erythropoiesis, chronic anemia, and iron overload contribute to the clinical complications of thalassemia intermedia through stepwise pathophysiological mechanisms. These complications, including splenomegaly, extramedullary erythropoiesis, iron accumulation, leg ulcers, thrombophilia, and bone abnormalities can be managed via fetal hemoglobin induction, occasional transfusions, chelation, and in some cases, stem cell transplantation. Given its clinical diversity, thalassemia intermedia patients require tailored approaches to therapy. Here we present an overview and novel approaches to the genetic basis, pathophysiological mechanisms, clinical complications, and optimal management of thalassemia intermedia.

Thiopurine S-Methyltransferase as a Pharmacogenetic Biomarker: Significance of Testing and Review of Major Methods.

BACKGROUND: Thiopurine S-methyltransferase (TPMT) enzyme metabolizes thiopurine drugs which are widely used in various disciplines as well as in leukemias. Individual enzyme activity varies depending on the genetic polymorphisms of TPMT gene located at chromosome 6. Up to 14% of population is known to have a decreased enzyme activity, and if treated with standard doses of thiopurines, these individuals are at a high risk of severe Adverse Drug Reactions (ADR) as myelosuppression, gastrointestinal intolerance, pancreatitis and hypersensitivity. However, TPMT-deficient patients can successfully be treated with decreased thiopurine doses if enzyme status is identified by a prior testing. TPMT status identification is a pioneering experience in application of pharmacogenetic testing in clinical settings. 4 TPMT (*2, *3A, *3B, *3C) alleles are known to account for 80-95% of a decreased enzyme activity, and therefore, identifying the presence of these alleles supported by phenotypic measurement of the enzyme activity can reveal patient's TPMT status. Evaluation of the levels of thiopurine metabolites further supports the practice of appropriate dose adjustment by providing the efficient monitoring of drug cytotoxicity. CONCLUSION: We hereby review the thiopurine pharmacogenetics and the methods applied in common practice to evaluate patient's TPMT status.

Genotype-Phenotype Correlations of ß-Thalassemia Mutations in an Azerbaijani Population.

ß-Thalassemia is the most common inherited disorder in Azerbaijan. The aim of our study was to reveal genotype-to-phenotype correlations of the most common ß-thalassemia mutations in an Azerbaijani population. Patients with codon 8 (-AA), IVS-I-6 (T>C), and IVS-II-1 (G>A) mutations, which are reportedly the most common ß-globin gene mutations among the local population, were tested for hematologic parameters. Fifty-five previously tested patients with known genotypes were included in the study. Hematologic indices and hemoglobin fractions were tested in order to reveal the phenotypic manifestation of the mutations. The results obtained indicate that clinical presentation varies between different ß-globin gene mutations: individuals with IVS-I-6 (T>C) mutations showed milder presentation than those with codon 8 (-AA) and IVS-II-1 (G>A), which is associated with the molecular basis of the mutations. These data can be of assistance to predict clinical presentation and select the best possible therapeutic approach via early genotype identification.

Identification of two rare ß-globin gene mutations in a patient with ß-thalassemia intermedia from Azerbaijan.

ß-Thalassemias are an inherited group of disorders of hemoglobin (Hb) and comprise the most common monogenic disorders in Azerbaijan. They are extremely heterogeneous at the molecular level. Here we report the first identification of a patient who is a compound heterozygote for two rare ß-thalassemia (ß-thal) mutations, IVS-I-130 (G>C) and codon 37 (TGG>TGA).