The Role of Caspase Genes Polymorphisms in Genetic Susceptibility to Philadelphia-Negative Myeloproliferative Neoplasms in a Portuguese Population.

Our main aim was to evaluate the role of caspases' genes SNPs in Philadelphia-chromosome negative chronic myeloproliferative neoplasms (PN-MPNs) susceptibility. A case-control study in 133 Caucasian Portuguese PN-MPNs patients and 281 matched controls was carried out, studying SNPs in apoptosis related caspases: rs1045485 and rs1035142 (CASP8), rs1052576, rs2308950, rs1132312 and rs1052571 (CASP9), rs2227309 and rs2227310 (CASP7) and rs13006529 (CASP10). After stratification by pathology diagnosis for essential thrombocythemia (ET), female gender or JAK2 positive, there is a significant increased risk for those carrying at least one variant allele for CASP9 (C653T) polymorphism (OR 2.300 CI 95% [1.180-4.484], P = 0.014). However, when considered individually, none of the studied caspases polymorphisms was associated with PN-MPNs risk. Our results do not reveal a significant involvement of caspase genes polymorphisms on the individual susceptibility towards PN-MPNs as a whole. However, for essential thrombocythemia (ET), female gender or JAK2 positive, there is a significant increased risk to those carrying at least one variant allele for CASP9. Although larger studies are required to confirm these results and to provide conclusive evidence of association between these and other caspases variants and PN-MPNs susceptibility, these new data may contribute to a best knowledge of the pathophysiology of these disorders and, in the future, to a more rational and efficient choice of therapeutic strategies to be adopted in PN-MPNs treatment.

Effects of polymorphic DNA genes involved in BER and caspase pathways on the clinical outcome of myeloproliferative neoplasms under treatment with hydroxyurea.

Several single nucleotide polymorphisms (SNPs) influencing DNA repair capacity and apoptotic status may confer genetic predisposition to Philadelphia­chromosome negative myeloproliferative neoplasms (PN­MPNs), and influence therapeutic response and the clinical course. In the present study, whether SNPs in genes involved in apoptosis and the base excision repair (BER) pathway was evaluated. In addition, some known risk factors in PN­MPNs that may influence survival and therapeutic response to hydroxyurea (HU) were analyzed, taking into account three items: Disease progression, predisposition to new non­myeloid neoplasms and thrombotic events. The present study involved a total of 133 Caucasian Portuguese PN­MPNs patients treated with HU, whereby 17 cases showed progression to myelofibrosis/leukemia, 11 developed new non­myeloid neoplasms and 22 presented with thrombotic events. Progression to secondary myelofibrosis/leukemia is influenced by exposure to cytoreductive agents, and caspase and BER polymorphisms {globally, CASP8 3'untranslated region [odds ratio (OR)=0.24; 95% confidence interval (CI), 0.08­0.69], XRCC1 Arg194Trp [OR=3.58; 95% CI, 0.98­13.01]; for essential thrombocythemia patients CASP9 Arg173His [OR=11.27; 95% CI, 1.13­112.28], APEX1 Asp148Glu [OR=0.28; 95% CI, 0.74­1.03], and XRCC1 Arg194Trp [OR=6.60; 95% CI, 1.60­27.06]}. Moreover, globally caspase and BER polymorphisms influenced the development of new nonmyeloid malignancies [CASP8 Asp270His (OR=5.90; 95% CI, 1.42­24.62) and XRCC1 Arg399Gln (OR=0.27; 95% CI, 0.07­1.03)]. On the other hand, only the BER pathway had a role in the presence of thrombotic events [XRCC1 Gln399Arg (OR=0.35; 95% CI, 0.14­0.88)]. JAK2 mutation had no influence on these complications. Larger studies are required to confirm these results, and to provide conclusive evidence of association between these and other variants with PN­MPNs therapeutic response and clinical evolution. However, this study may allow the development of drugs more directly targeted to the pathophysiology of the disease, with high efficacy, fewer adverse effects, contributing to compliance of patients with treatments. The clinical indication for classical drugs, including HU, may be guided by variant genes, which may provide additional beneficial effects.

Prevalence of the Janus kinase 2 V617F mutation in Philadelphia-negative myeloproliferative neoplasms in a Portuguese population.

Myeloproliferative neoplasms (MPNs) result from the malignant transformation of a hematopoietic stem-cell (HSC), leading to abnormal amplification and proliferation of myeloid lineages. Identification of the Janus kinase 2 (JAK2) V617F mutation developed the knowledge of Philadelphia-negative (PN)-MPNs, contributing to and influencing the definition of the phenotype and prognostic impact. Considering the lack of Portuguese epidemiological data, the present study intends to characterize the prevalence of the JAK2 mutation in a PN-MPN versus a control Portuguese population. Caucasian Portuguese PN-MPN patients (n=133) and 281 matched control subjects were investigated. No significant differences were identified between the case and control groups concerning age distribution or smoking habits. Pathology distribution was as follows: 60.2% with essential thrombocythemia (ET), 29.3% with polycythemia vera (PV) and 10.5% with primary myelofibrosis (PMF). A total of 75.0% of patients were positive for the presence of the JAK2 V617F mutation. In addition, the prevalence of PV was 87.2%, ET was 73.4% and PMF was 50.0%. The JAK2 V617F mutation is observed in various MPN phenotypes, and has an increased incidence in ET patients and a decreased incidence in PV patients. These data may contribute to improving the knowledge of the pathophysiology of these disorders, and to a more rational and efficient selection of therapeutic strategies to be adopted, notably because most of the patients are JAK2 V617F negative.

DNA repair genes polymorphisms and genetic susceptibility to Philadelphia-negative myeloproliferative neoplasms in a Portuguese population: The role of base excision repair genes polymorphisms.

The role of base excision repair (BER) genes in Philadelphia-negative (PN)-myeloproliferative neoplasms (MPNs) susceptibility was evaluated by genotyping eight polymorphisms [apurinic/apyrimidinic endodeoxyribonuclease 1, mutY DNA glycosylase, earlier mutY homolog (E. coli) (MUTYH), 8-oxoguanine DNA glycosylase 1, poly (ADP-ribose) polymerase (PARP) 1, PARP4 and X-ray repair cross-complementing 1 (XRCC1)] in a case-control study involving 133 Caucasian Portuguese patients. The results did not reveal a correlation between individual BER polymorphisms and PN-MPNs when considered as a whole. However, stratification for essential thrombocythaemia revealed i) borderline effect/tendency to increased risk when carrying at least one variant allele for XRCC1_399 single-nucleotide polymorphism (SNP); ii) decreased risk for Janus kinase 2-positive patients carrying at least one variant allele for XRCC1_399 SNP; and iii) decreased risk in females carrying at least one variant allele for MUTYH SNP. Combination of alleles demonstrated an increased risk to PN-MPNs for one specific haplogroup. These findings may provide evidence for gene variants in susceptibility to MPNs. Indeed, common variants in DNA repair genes may hamper the capacity to repair DNA, thus increasing cancer susceptibility.

Chemokine receptor repertoire reflects mature T-cell lymphoproliferative disorder clinical presentation.

The World Health Organization classification of mature T-cell lymphoproliferative disorders, combines clinical, morphological and immunophenotypic data. The latter is a major contributor to the classification, as well as to the understanding of the malignant T-cell behavior. The fact that T-cell migration is regulated by chemokines should, in theory, enable us to identify tissue tropism and organ involvement by neoplastic T-cells by monitoring chemokine receptor surface expression. To address this issue we compared the expression of several early and late inflammatory, homeostatic, and organ specific chemokine receptors on blood T-cells from normal individuals and patients with T-cell large granular lymphocytic leukemia and peripheral T-cell lymphoma. T-cell large granular lymphocytic leukemia cells mainly express late inflammatory chemokine receptors (CXCR1 and CXCR2), whereas peripheral T-cell lymphoma cells usually express one or more organ homing receptors (CCR4, CCR6 and CCR7). Nevertheless, no clear correlation was found between CCR4 and CCR7 expression and skin and lymph node involvement, respectively. Compared to their normal counterparts, lymphoma T-cells displayed an exaggerated CCR4 expression, whereas leukemic T-cells had abnormally high CXCR1 and CXCR2 expression. Further analysis revealed that, in leukemia patients, the percentage of neoplastic cells expressing CCR5 correlates directly with lymphocytosis. In addition, in the case of CD8 T-cell leukemia patients, an inverse correlation with neutropenia was found. In lymphoma patients, higher CCR4 and CCR7 expression is accompanied by lower to absent CCR5 expression.

Combined molecular diagnosis of B-cell lymphomas with t(11;14)(q13;q32) or t(14;18)(q32;q21) using multiplex- and long distance inverse-polymerase chain reaction.

Translocations t(14;18)(q32;q21) and t(11;14)(q13;q32) are recurrent findings in follicular lymphoma (FL) and mantle cell lymphoma (MCL), respectively. However, the molecular counterparts of these translocations can only be detected in up to 75% of FL and 50% of MCL cases using routine techniques. To improve the efficiency of detection, we first devised a single-tube multiplex-polymerase chain reaction (PCR) assay with primers located within a conserved immunoglobulin heavy chain (IGH) sequence and 5' to the main breakpoint cluster regions of BCL2 and CCND1. Using this assay in 17 FL and 11 MCL diagnostic DNA samples, we readily identified a BCL2-IGH fusion in 65% of FL patients and a CCND1-IGH fusion in 55% of MCL patients. In the remaining cases, we used long distance inverse-PCR to detect BCL2-IGH and CCND1-IGH fusion genes with different BCL2 and CCND1 breakpoint locations. We found additional translocations in 3 patients (17%) with FL and in 4 patients (36%) with MCL. Taken together, we show that multiplex-PCR combined with long distance inverse-PCR detected a t(14;18) in 82% of FL patients and a t(11;14) in 91% of MCL patients, demonstrating that this 2-step protocol is an effective approach for molecular detection of t(11;14) and t(14;18) in B-cell lymphomas.

Asymptomatic homozygous deletional beta(0)-thalassemia in an African individual.

Homozygosity or compound heterozygosity for beta(0)-thalassemia mutations most commonly results in a transfusion-dependent thalassemia major phenotype. In this report, we describe a 55-year-old male, from Guinea-Bissau, that had been asymptomatic and never transfused until being admitted to hospital with anemia, fever, splenomegaly, and asthenia. Following hospital admission, HIV-2 and Mycobacterium tuberculosis infections were diagnosed, and biochemical and molecular studies revealed homozygosity for beta(0)-thalassemia. At the molecular level, this is the first description of homozygosity for the beta(0)-Black 1,393-bp deletion. In this case, the complete absence of beta-globin gene expression seems to be compensated by an unusually high fetal globin gene expression (Hb F 96%). Beta-globin haplotyping results were compatible with the propositus being homozygous for the Black 2 haplotype and for the absence of the XmnI polymorphism at -158 of (G)gamma-globin gene (-/-). Co-inheritance of genetic factors usually associated with high Hb F levels was not detected. Otherwise, the propositus is a heterozygote for the alpha-globin gene 3.7-kb deletion that is a beneficial modulating factor but not sufficient to explain this extremely mild phenotype. This unusual genotype/phenotype association is discussed in terms of the mechanisms underlying hemoglobin switching during development.

Fetal hemoglobin elevation in Hb Lepore heterozygotes and its correlation with beta globin cluster linked determinants.

We have analysed, at the hematological and molecular level, 51 Hb lepore heterozygotes and three compound heterozygotes for Hb Lepore and HbS (HbLep/HbS) in 26 unselected Portuguese families. The Lepore Boston variant was present in one family, in association with classical haplotype V. All of the other Lepore alleles present haplotype III in association with XmnI (+)5' of (G)gamma gene, in tight linkage disequilibrium to the major mutation found in the Portuguese population, the Lepore Baltimore variant ( delta(68Leu)-beta(84Thr)). The three compound heterozygotes are the first HbLep/HbS individuals reported in the literature, with the Lepore Baltimore mutation linked to haplotype III. In agreement with other studies, these Lepore Baltimore heterozygotes have higher HbF (1.4-14.1% of total hemoglobin) than published cases of Lepore Boston (0.8-5.4%), which is associated with XmnI(-). Among the Lepore Baltimore heterozygotes, the (AT)xTy repeat region at -540 bp of the beta globin gene in trans to the Lepore chromosome, can account for much of the variability in HbF level. The allele (AT)7T7 is associated with lower HbF, and (AT)9T5 is associated with higher HbF. As we previously reported for beta thalassemic carriers, we observe in Lepore Baltimore carriers an effector in trans, linked to the (AT)xTy sequence, acting as an HPFH (Hereditary Persistence of Fetal Hemoglobin) determinant.