Thromboembolic events in patients with paroxysmal nocturnal hemoglobinuria (PNH): Real world data of a Greek nationwide multicenter retrospective study.

Thrombosis is the most common and a life-threatening complication in patients with Paroxysmal Nocturnal Hemoglobinuria. One-third of patients with PNH experience at least one thromboembolic event during the course of the disease, with thrombosis being the most common cause of death in these patients. The mechanism of thrombosis in PNH is complex and continues to be of great research interest. Since the introduction of C5 complement inhibitors in the treatment of PNH, the incidence of thromboembolic events has decreased substantially. We retrospectively analyzed data concerning the thrombotic episodes of 41 patients with PNH from 14 different national hematology centers in Greece. Sixteen patients (39%) experienced at least one episode of thrombosis, including, seven (43.8%) at diagnosis, seven (43.8%) during the course of the disease and two (12.5%) patients prior to PNH diagnosis. Nearly half of these individuals (n=7, 43.8%) had multiple episodes of thrombosis during the course of their disease. The most common sites of thrombosis were intra-abdominal veins. Three out of 26 patients developed thrombosis while on eculizumab. In none of the 16 patients, the thrombotic event was fatal. Our findings, despite the small number of patients, confirmed that thrombosis continues to be a significant complication of PNH affecting more than one third of the patients.

ETV6/RUNX1-positive childhood acute lymphoblastic leukemia (ALL): The spectrum of clonal heterogeneity and its impact on prognosis.

The prognostic significance of the ETV6/RUNX1-fusion and of the accompanying aberrations is disputable; whether co-existing sub-clones are responsible for delayed MRD-clearance and thus, moderate outcome, remains to be clarified. We studied, in a paediatric cohort of 119 B-ALLs, the relation between the ETV6/RUNX1 aberration and the co-existing subclones with (a) presenting clinical/biological features, (b) early response to treatment(MRD) and (c) long-term outcome over a 12-year period. Patients were homogeneously treated according to BFM-based-protocols. 27/119 patients (22.7%) were ETV6/RUNX1-positive; 19/27 (70.4%) harbored additional genetic abnormalities while 9/19 (33.3%) presented with clonal heterogeneity. The most common abnormalities were del12p13 (37%), 3-6×21q22 (22.2%), del9p21 (18.5%) and 2-3xETV6/RUNX1 (18.5%). MRDd15-positivity (≥10-3) was detected in 44% of the cohort; the corresponding MRD among patients carrying subclones rises to 88.9%. Common features of all relapses were sub-clonal diversity, FCM-MRDd15-positivity and additional del(9p21) while there were no censored relapses among ETV6/RUNX1-positive patients with sole translocation and absence of additional aberrations, within a median follow-up time of 90 months. In our study, the presence of clonal heterogeneity and impaired FCM-MRD clearance among ETV6/RUNX1-positive patients, ultimately influenced prognosis. Longer follow-up is needed in order to further validate these initial results.

Polymorphisms and haplotypes in MyD88 are associated with the development of sarcoidosis: a candidate-gene association study.

Sarcoidosis is considered as a disorder of protracted immune response to an as yet unidentified causative agent that leads to granuloma formation. Material from M. tuberculosis and P. acne has been repeatedly detected in the sarcoidosis lesions, implying the involvement of the Toll-like receptor2 (TLR2) gene that responds to these intracellular pathogens. Since TLR2 association studies have produced controversial results, we sought to investigate whether the downstream signalling molecule MyD88 could be linked to disease susceptibility. We analyzed a total of 93 cases with sarcoidosis and of 89 controls for the most common MyD88 SNPs: -938C>A (rs4988453) and 1944C>G (rs4988457). There is evidence that the genotype distributions of both variants are associated with the development of sarcoidosis (p = 0.038 for -938C>A and p = 0.026 for 1944C>G). In particular, -938A and 1944G carriers were associated with risk of sarcoidosis [OR = 2.48 (1.23-5.02) and OR = 0.33 (0.14-0.76)], respectively, indicating dominance of the mutant alleles; however, the adjustment of the effect size for age and sex diminished the significance. The haplotype analysis showed association for the -938A/1944G haplotype (p < 0.001). Since genetic association studies have linked MyD88 to Hodgkin's lymphoma it is tempting to speculate that MyD88 may contribute to the granuloma formation that characterizes sarcoidosis.

Comparative analysis of FV vectors with human α- or ß-globin gene regulatory elements for the correction of ß-thalassemia.

ß-Globin locus control region (LCR) sequences have been widely used for the regulated expression of the human ß-globin gene in therapeutic viral vectors. In this study, we compare the expression of the human ß-globin gene from either the HS2/HS3 ß-globin LCR or the HS40 regulatory element from the α-globin locus in the context of foamy virus (FV) vectors for the genetic correction of ß-thalassemia. Both regulatory elements expressed comparable levels of human ß-globin in a murine erythroleukemic line, whereas in murine hematopoietic stem cells the HS40.ß vector proved more efficient in ß-globin expression and correction of the ß-thalassemia phenotype. Following transplantation in the Hbb(th3/+) mouse model, the expression efficiency by the two vectors was similar, whereas the HS40.ß vector achieved relatively more stable transgene expression. In addition, in an ex vivo assay using CD34+ cells from thalassemic patients, both vectors achieved significant human ß-globin expression and restoration of the thalassemic phenotype as evidenced by enhanced erythropoiesis and decreased apoptosis. Our data suggest that FV vectors with the α-globin HS40 element can be used as alternative but equally efficient vehicles for human ß-globin gene expression for the genetic correction of ß-thalassemia.

Successful control of acute myelofibrosis with lenalidomide.

Acute panmyelosis with myelofibrosis (APMF) is a rare, fatal hematological neoplasm that is characterized by the acute onset of cytopenias and fibrosis in the bone marrow in the absence of splenomegaly or fibrosis-related morphological changes in the RBCs. We present the case of a 59-year-old female who presented with a two-month history of anemia, leucopenia and a normal platelet count. The marrow was heavily fibrotic, and no aspirate material could be obtained; the biopsy showed extensive infiltration with small to medium size megakaryocytes, dysplastic changes in the erythroid compartment, and left shift in the myeloid cells. The patient was treated for four months with anabolic steroids (Danazol), growth factors and received regular blood transfusions. At 4 months after diagnosis, the patient was started on Lenalidomide, 10 mg/day for a 21-d-course along with growth factor support. At 6 months after treatment, the patient was transfusion-independent, had normalized blood counts, and, at 32 months on continuous lenalidomide treatment, her needs for growth factor support have been minimized. Repeat bone marrow biopsies showed a patchy distribution of fibrosis with areas of normal cellularity and morphology. To our knowledge, this is the first case for a medication that could reverse the fatal outcome of APMF.

Dual transgene expression by foamy virus vectors carrying an endogenous bidirectional promoter.

Several gene therapy applications require the transfer and simultaneous expression of multiple genes in the same cell. In this study, we analyzed the potential for coordinated expression of an endogenous bidirectional promoter located on chromosome X, which controls the expression of the heterogeneous nuclear ribonucleoprotein H2 (HNRNPH2) and alpha-galactosidase (GLA) genes. The promoter was cloned in both transcriptional orientations in a foamy virus (FV) vector backbone, whereas the enhanced green fluorescent protein (EGFP) and low-affinity nerve growth factor receptor (DeltaLNGFR) reporter genes were cloned in the 5'-3' and 3'-5' transcriptional orientations, respectively. In all the cell lines tested, both vectors showed high levels of transgene coexpression that reached 76% of total positive cells (range from 76 to 18%). Comparison of EGFP and DeltaNGFR levels revealed that the side of the promoter that drives the expression of the HNRNPH2 gene in the genome was stronger and in accordance to its in situ activity. When tested with CD34(+) cells, transgene coexpression reached 35.3% of all positive cells in progenitor assays and 16.8% of all positive cells after transplantation in NOD/severe combined immunodeficient mice. In summary, we show that the endogenous promoter used in this study holds bidirectional activity in the context of FV vectors and can be used in gene therapy applications requiring synchronized expression of two genes.

Gene transfer into murine hematopoietic stem cells with helper-free foamy virus vectors.

Gene transfer into hematopoietic stem cells (HSCs) is an ideal treatment strategy for many genetic and hematologic diseases. However, progress has been limited by the low HSC transduction rates obtained with retroviral vectors based on murine leukemia viruses. This study examined the potential of vectors derived from the nonpathogenic human foamy virus (HFV) to transduce human CD34(+) cells and murine HSCs. More than 80% of human hematopoietic progenitors present in CD34(+) cell preparations derived from cord blood were transduced by a single overnight exposure to HFV vector stocks. Mice that received transduced bone marrow cells expressed the vector-encoded transgene long term in all major hematopoietic cell lineages and in over 50% of cells in some animals. Secondary bone marrow transplants and integration site analysis confirmed that gene transfer occurred at the stem cell level. Transgene silencing was not observed. Thus vectors based on foamy viruses represent a promising approach for HSC gene therapy. (Blood. 2001;98:604-609)

Novel in vitro assay for the detection of pharmacologic inducers of fetal hemoglobin.

Current techniques for identifying fetal hemoglobin (HbF) inducers are complex and time consuming. We developed a rapid and efficient method for detecting HbF inducers. Our system uses a recombinant DNA construct in which the coding sequences of 2 different luciferase reporter genes, firefly and renilla, are substituted for those of human gamma and beta globin genes, respectively. The activity of these genes can be distinguished by a simple, highly sensitive enzymatic assay in cell lysates. GM979 cells stably transfected with the construct are cultured in the presence of compounds, and their effects are determined by measuring the changes in activity of the 2 luciferase genes. Specific gamma globin gene inducers are recognized by their ability to increase gamma-firefly luciferase (gamma(F)) gene activity significantly more than beta-renilla luciferase (beta(R)) gene activity, identified by an increased ratio of gamma-firefly luciferase activity over total luciferase activity. These results suggest that the use of the 2 luciferase reporter genes provides a simple, highly sensitive, and reproducible system for the detection of compounds that increase gamma-globin gene expression. It can therefore be used for the screening of chemical agents that may have gamma-globin gene inducibility.

Correct function of the locus control region may require passage through a nonerythroid cellular environment.

The function of the beta-globin locus control region (LCR) has been studied both in cell lines and in transgenic mice. We have previously shown that when a 248-kb beta-locus YAC was first microinjected into L-cells and then transferred into MEL cells by fusion, the YAC loci of the LxMEL hybrids displayed normal expression and developmental regulation.To test whether direct transfer of a beta-globin locus (beta-YAC) into MEL cells could be used for studies of the function of the LCR, a 155-kb beta-YAC that encompasses the entire beta-globin locus was used. This YAC was retrofitted with a PGK-neo selectable marker and with two I-PpoI sites at the vector arm-cloned insert junctions, allowing detection of the intact globin loci on a single I-PpoI fragment by pulsed field gel electrophoresis (PFGE). The Ppo-155 beta-YAC was used to directly lipofect MEL 585 cells. In 7 beta-YAC MEL clones with at least one intact copy of the YAC, the levels of total human globin mRNA (ie, epsilon + gamma + beta) per copy of integrated beta-YAC varied more than 97-fold between clones. These results indicated that globin gene expression was strongly influenced by the position of integration of the beta-YAC into the MEL cell genome and suggested that the LCR cannot function properly when the locus is directly transferred into an erythroid cell environment as naked beta-YAC DNA. To test whether passage of the beta-YAC through L-cells before transfer into MEL cells was the reason for the previously observed correct developmental regulation of human globin genes in the LxMEL hybrid cells, we transfected the YAC into L-cells by lipofection. Three clones carried the intact 144-kb I-PpoI fragment and transcribed the human globin genes with a fetal-like pattern. Subsequent transfer of the YAC of these L(beta-YAC) clones into MEL cells by fusion resulted in LxMEL hybrids that synthesized human globin mRNA. The variation in human beta-globin mRNA (ie, epsilon + gamma + beta) levels between hybrids was 2.5-fold, indicating that globin gene expression was independent of position of integration of the transgene, as expected for normal LCR function. The correct function of the LCR when the YAC is first transferred into the L-cell environment raises the possibility that normal activation of the LCR requires interaction with the transcriptional environment of an uncommitted, nonerythroid cell. We propose that the activation of the LCR may represent a multistep process initiated by the binding of ubiquitous transcription factors early during the differentiation of hematopoietic stem cells and completed with the binding of erythroid type of factors in the committed erythroid progenitors.

Comparison of expression of human globin genes transferred into mouse erythroleukemia cells and in transgenic mice.

To examine whether transfer of gamma globin genes into mouse erythroleukemia cells can be used for the analysis of regulatory elements of gamma globin gene promoter, Agamma gene constructs carrying promoter truncations that have been previously analyzed in transgenic mice were used for production of stably transfected mouse erythroleukemia (MEL) cell clones and pools. We found that constructs, which contain a microlocus control region (microLCR) that efficiently protects globin gene expression from the effects of the position of integration in transgenic mice, display position-dependent globin gene expression in MEL cell clones. Agamma globin gene expression among MEL cell clones carrying the muLCR(-201)Agamma and muLCR(-382)Agamma gene constructs ranged 15.5-fold and 17.6-fold, respectively, and there was no correlation between the Agamma mRNA levels and the copies of the transgene (r = .28, P = .18). There was significant variation in per copy Agamma globin gene expression among MEL cell pools composed of 10 clones, but not among pools composed of 50 clones, indicating that position effects are averaged in pools composed by large numbers of clones. The overall pattern of Agamma globin gene expression in MEL cell pools resembled that observed in transgenic mice indicating that MEL cell transfections can be used in the study of cis elements controlling gamma globin gene expression. MEL cell transfections, however, are not appropriate for investigation of cis elements, which either sensitize or protect the globin transgenes from position effects.

Hb Arta [beta 45 (CD4) Phe-->Cys]: a new unstable haemoglobin with reduced oxygen affinity in trans with beta-thalassaemia.

The interaction of rare Hb variants with beta(0)-thalassaemia results in a quasihomozygous state where the erythrocytes contain the variant as the only major adult Hb component. Such a situation is a unique model that enables functional studies even in the case of a neutral variant that could not be isolated from Hb A. We report here an unusual patient carrying Hb Arta, a novel Hb variant [beta 45 (CD4) Phe-->Cys], in trans with beta(0)-thalassaemia gene (beta(0) 39). The aminoacid substitution at the critical CD corner of this Hb molecular renders the molecule unstable. In addition, haem is displaced in a position that favours the deoxy (T) conformation of the variant, but less than in Hb Cheverly [beta 45 (CD4) Phe-->Ser], and results in a p50 of 43 mmHg (pH 7.4, 37 degrees C) in the red cells with preservation of cooperativity. Solution studies of the almost pure Hb Arta show a 50% decrease in oxygen affinity and normal cooperativity; the Bohr effect and the interaction with organic phosphates are similar to those of Hb A. Hb Arta retains both normal homo- and heterotropic effects allowing a well-preserved oxygen transport in vivo despite a mild anaemia.

Pulmonary hypertension and right heart failure in patients with beta-thalassemia intermedia.

We analyzed seven patients with beta-thalassemia intermedia presenting with congestive heart failure secondary to pulmonary hypertension. This condition has been recognized only recently as part of the clinical spectrum of beta-thalassemia. Our group of patients included two men and five women with the clinical picture and laboratory data typical of beta-thalassemia intermedia. The mean age was 37.7 +/- 11.4 years, mean hematocrit value was 28.5 +/- 1.8%, mean number of transfused blood units was 171 +/- 153, and mean serum ferritin levels were 4,428 +/- 2,006 ng/mL. All but one of these patients had undergone splenectomy. Common findings of the investigative procedures include the following: dilation of the main pulmonary artery and cardiac enlargement in the chest radiograph; signs of right ventricular hypertrophy in the ECG; and dilated right ventricle with good left ventricular function in the echo study. Right heart catheterization showed the pulmonary systolic pressure to range from 55 to 90 mm Hg (74.1 +/- 10.3), pulmonary diastolic pressure from 25 to 50 mm Hg (37.7 +/- 8.7), mean pressure from 35 to 60 mm Hg (49.7 +/- 7.9), and pulmonary vascular resistance from 267 to 667 dynes.s.cm-5. Pulmonary capillary wedge pressure was within the normal range of values. The pathophysiologic condition of pulmonary hypertension in these patients is most probably associated with beta-thalassemia. There are mechanisms that increase cardiac output and at the same time restrict the pulmonary vascular bed. The results of this study imply that treatment decisions should be reconsidered for such patients.

Angioid streaks in sickle-thalassemia.

Angioid streaks have been described in a diverse group of diseases including hemoglobinopathies such as sickle cell anemia and beta-thalassemia. We investigated the prevalence of angioid streaks and pseudoxanthoma elasticum in the rare situation of patients who had compound heterozygous traits for hemoglobin S and beta-thalassemia. We examined 58 consecutive patients with sickle-thalassemia. Of these, 25 were men and 33 were women, and they ranged in age from 19 to 58 years (mean, 32.6 years). Angioid streaks were identified in six of 58 patients (10%), and of these three also displayed the cutaneous lesions of pseudoxanthoma elasticum, which were confirmed by skin biopsy. An expanded study on several relatives of the patients with angioid streaks failed to identify any similar cases. Statistical evaluation of the main hematologic and biochemical parameters in the patients with and without angioid streaks did not demonstrate any significant differences, except that the thalassemic component in all six patients with angioid streaks was beta(0) (that is, did not allow the synthesis of hemoglobin A). We conclude that angioid streaks and pseudoxanthoma elasticum skin lesions occur with an increased frequency in patients with sickle-thalassemia.

Molecular characterization of beta-thalassemia in Egyptians.

We sought to determine the spectrum of mutations producing beta-thalassemia in Egypt using genomic PCR and a variety of mutation-screening procedures. Thirty-four beta-thalassemia and three Hb S/beta-thalassemia patients originating from different regions of Egypt were studied, and the causative mutation was found in 69 of 71 (97%) beta-thalassemia genes. Four mutations accounted for 78% of beta-thalassemia genes in this population; IVS-1, nt 110 (41%), IVS-1 nt 6 (13%), IVS-1, nt 1 (13%), and IVS-2, nt 848 (11%). The latter allele, a C-A mutation at the third nucleotide of an acceptor site consensus sequence, has been described previously only in one Egyptian, one Iranian, one Tunisian, and one Black American patient. Nine other alleles each accounted for 1-3% of beta-thalassemia genes. Among these was one codon 27 allele (Hb Knossos), two frameshift 106/107 alleles previously seen only in a Black American, and a rarely observed mutation in the distal promoter region of the beta-globin gene, -87 (C-A). Our results suggest that from a molecular genetic standpoint a beta-thalassemia prevention program based on carrier screening and prenatal diagnosis can be implemented in Egypt. In couples at risk for beta-thalassemia, the causative mutation should be identifiable in both members in 92% and in one member in the remaining 8%.