Severe methylenetetrahydrofolate reductase deficiency revealed by a pulmonary embolism in a young adult

Deficiency in methylenetetrahydrofolate reductase (MTHFR), the enzyme involved in the remethylation of homocysteine to methionine using methyltetrahydrofolate as cofactor, induces hyperhomocysteinaemia, homocysteinuria, hypomethioninaemia and low methylfolate levels. Diagnosis usually occurs during infancy because of various neurological abnormalities. We report MTHFR deficiency diagnosed in an adult woman after a pulmonary embolism. Her adult sister, intellectually retarded, suffered from the same disease. Molecular analysis of the MTHFR gene exhibited four different mutations (two missense mutations, one exon skipping and C677T). The impact of these mutations was analysed through the biological abnormalities in the parents and children.     

Should transcobalamin deficiency be treated aggressively?

Transcobalamin (transcobalamin II, TC) transports plasma vitamin B₁₂ (cobalamin, Cbl) into cells. TC deficiency is a rare autosomal recessive disorder causing intracellular Cbl depletion, which in turn causes megaloblastic bone marrow failure, accumulation of homocysteine and methylmalonic acid, and methionine depletion. The clinical presentation reflects intracellular Cbl defects, with early-onset failure to thrive with gastrointestinal symptoms, pancytopenia, and megaloblastic anemia, sometimes followed by neurological complications. We report the clinical, biological, and molecular findings and the outcome in five TC-deficient patients. The three treated early had an initial favorable outcome, whereas the two treated inadequately had late-onset severe neuro-ophthalmological impairment. Even if the natural course of the disease over time might also result in late-onset symptoms in the aggressively treated patients, these data emphasize that TC deficiency is a severe disorder requiring early detection and probably long-term aggressive therapy. Mutation analysis revealed six unreported mutations in the TCN2 gene. In silico structural analysis showed that these mutations disrupt the Cbl-TC interaction domain and/or the putative transcobalamin–transcobalamin receptor interaction domain.     

Proteasome inhibitor bortezomib impairs both myelofibrosis and osteosclerosis induced by high thrombopoietin levels in mice

Primary myelofibrosis (PMF) is the most serious myeloproliferative disorder, characterized by clonal myeloproliferation associated with cytokine-mediated bone marrow stromal reaction including fibrosis and osteosclerosis. Current drug therapy remains mainly palliative. Because the NF-kappaB pathway is implicated in the abnormal release of cytokines in PMF, the proteasome inhibitor bortezomib might be a potential therapy. To test its effect, we used the lethal murine model of myelofibrosis induced by thrombopoietin (TPO) overexpression. In this TPO(high) model, the development of the disease is related to a deregulated MPL signaling, as recently described in PMF patients. We first demonstrated that bortezomib was able to inhibit TPO-induced NF-kappaB activation in vitro in murine megakaryocytes. It also inhibited NF-kappaB activation in vivo in TPO(high) mice leading to decreased IL-1alpha plasma levels. After 4 weeks of treatment, bortezomib decreased TGF-beta1 levels in marrow fluids and impaired marrow and spleen fibrosis development. After 12 weeks of treatment, bortezomib also impaired osteosclerosis development through osteoprotegerin inhibition. Moreover, this drug reduced myeloproliferation induced by high TPO level. Finally, bortezomib dramatically improved TPO(high) mouse survival (89% vs 8% at week 52). We conclude that bortezomib appears as a promising therapy for future treatment of PMF patients.     

Isolated remethylation disorders: do our treatments benefit patients?

Deficiency of 5,10-methylenetetrahydrofolate reductase (MTHFR), the very rare methionine synthase reductase (CblE) and methionine synthase (CblG) defects, and the recently identified CblD-variant-1 defect are primary remethylation defects characterized by an isolated defect in methionine synthesis without methylmalonic aciduria. The clinical signs are mainly neurological, and hematological signs are seen in CblE, CblG, and CblD-variant-1 defects. Patients with neonatal or early-onset disease exhibit acute neurological distress. Infants and children have unspecific mental retardation, often with acquired microcephaly. Without appropriate therapy, they may experience acute or rapidly progressive neurological deterioration, which may be fatal. Adolescents and adults show normal development or mild developmental delay initially and then experience rapid neurological or behavioral deterioration. A few patients may have signs of subacute combined degeneration of the spinal cord. Adults may be asymptomatic or present with isolated thromboembolism. All patients with suspected remethylation disorders should receive emergency treatment with parenteral administration of hydroxocobalamin and folate supplements combined with betaine orally. The long-term treatment of CblE, CblG, and CblD-variant-1 defects consists of parenterally administered hydroxocobalamin and orally administered folate and betaine supplements, whereas patients with MTHFR deficiency require long-term oral folate and betaine supplements. Long-term oral methionine therapy should also be considered. Early treatment may lead to a favorable outcome with developmental recovery and prevention of further neurological deterioration. In contrast, most late-treated patients have severe and irreversible neuromotor impairments. Hematological abnormalities are easily corrected.     

Overexpression of FKBP51 in idiopathic myelofibrosis regulates the growth factor independence of megakaryocyte progenitors

Idiopathic myelofibrosis (IMF) is a chronic myeloproliferative disorder characterized by megakaryocyte hyperplasia and bone marrow fibrosis. Biologically, an autonomous megakaryocyte growth and differentiation is noticed, which contributes to the megakaryocyte accumulation. To better understand the molecular mechanisms involved in this spontaneous growth, we searched for genes differentially expressed between normal megakaryocytes requiring cytokines to grow and IMF spontaneously proliferating megakaryocytes. Using a differential display technique, we found that the immunophilin FKBP51 was 2 to 8 times overexpressed in megakaryocytes derived from patients' CD34(+) cells in comparison to normal megakaryocytes. Overexpression was moderate and confirmed in 8 of 10 patients, both at the mRNA and protein levels. Overexpression of FKBP51 in a UT-7/Mpl cell line and in normal CD34(+) cells induced a resistance to apoptosis mediated by cytokine deprivation with no effect on proliferation. FKBP51 interacts with both calcineurin and heat shock protein (HSP)70/HSP90. However, a mutant FKBP51 deleted in the HSP70/HSP90 binding site kept the antiapoptotic effect, suggesting that the calcineurin pathway was responsible for the FKBP51 effect. Overexpression of FKBP51 in UT-7/Mpl cells induced a marked inhibition of calcineurin activity. Pharmacologic inhibition of calcineurin by cyclosporin A mimicked the effect of FKBP51. The data support the conclusion that FKBP51 inhibits apoptosis through a calcineurin-dependent pathway. In conclusion, FKBP51 is overexpressed in IMF megakaryocytes and this overexpression could be, in part, responsible for the megakaryocytic accumulation observed in this disorder by regulating their apoptotic program.     

Distribution of calcineurin activity in blood cell fractions and impact of tacrolimus inhibition

The aim of this study was to investigate calcineurin (PP2B) activity in different blood cell fractions and its inhibition by tacrolimus. Basal PP2B activity was measured in each blood cellular fraction collected from healthy volunteers. The inhibition profile of PP2B activity was explored in isolated peripheral blood mononuclear cells (PBMC) and platelets exposed directly to tacrolimus and in PBMC and platelets isolated from whole blood previously exposed to tacrolimus. Contrasting with red blood cells (30%) and platelets (25%), PBMC represented only 8.7% of PP2B activity of unfractionated whole blood. After tacrolimus exposure of isolated PBMC and platelets, the concentration of tacrolimus required to inhibit 50% of PP2B activity (EC(50)) in PBMC was significantly lower than in platelets (0.26 ng/mL vs. 0.83 ng/mL, P < 0.001). EC(50) values were similar in PBMC and platelets isolated from whole blood previously exposed to tacrolimus (7.69 ng/mL vs. 7.42 ng/mL, respectively). These results suggest PBMC is a very suitable matrix for PP2B measurement in monitoring transplant recipients but clinical studies are necessary to solve clearly this issue.     

Removal of surface by-products from sintered hydroxyapatite: effect of a chelation treatment on fibronectin adsorption and cell adhesion

It was observed that fibronectin precipitates when deposited on hydroxyapatite (HA) ceramics. Fibronectin's known affinity for calcium and the composition of the ceramic itself suggested that calcium release could be the main cause of this aggregation effect. It was then decided to investigate the effect of a surface chelation treatment on fibronectin adsorption, and MG63 cell adhesion, onto porous ceramics of hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), and HA/TCP biphasic material (BCP). Those ceramics were immersed in an EDTA solution and the effect of this treatment on the material composition was assayed. X-ray diffraction data showed the presence of alpha- and beta-TCP phases in HA and BCP materials, which were both completely removed by the chelation treatment in the case of HA. On BCP, alpha-TCP was removed and beta-TCP partially dissolved. The TCP material, which was pure beta-TCP, underwent a mass loss, but no change in composition was observed. Adhesion of MG63 cells was overall higher on the fibronectin-coated EDTA-treated HA material, but was especially enhanced on EDTA-treated HA. Changes in surface morphologies, as compared with the use of scanning electron microscopy, did not seem to be related to the effects observed. The EDTA treatment proved to be a very efficient way of removing by-products of HA sintered materials, and thus enhancing the biocompatibility of the material.     

Dysregulated Mpl-Ligand production by hemopoietic cellsinduces a fatal myeloproliferative syndrome in mice

To evaluate the effects of long-term high-dose exposure to Mpl-ligand also called thrombopoietin (TPO), C57BL/6J murine marrow cells were infected with a retrovirus carrying the murine TPO gene. Mice were treated 4 days by 5-FU and marrow cells were then infected by coculture using a MPZen vector containing the murine TPO cDNA. Non adherent marrow cells were transplanted into lethally irradiated recipients. A majority of hematopoietic cells in the marrow, spleen, thymus and blood was transduced by the retroviral vector, one and three months after reconstitution. Plasma TPO activity in transplanted mice was extremely high (10⁴ U/ml). A disease with two distinct steps was observed. During the two first months after transplantation, platelet (plt) and white blood cell (WBC) counts increased 4- and 10-fold, respectively. Abnormal platelet size and granules were observed. Spleen weight increased 4-fold and marrow cellularity decreased 5-fold. Histology revealed hyperplasia of the megakaryocytic and myeloid cells. Total numbers of CFU-MK and CFU-GM increased. In contrast, the hematocrit progressively fell accompanied by a decrease in the erythroblasts and CFU-E numbers. Beginning two months after transplantation, plt and WBC numbers also declined. Thrombocytopenia was noted 5 months after transplantation. The Hcts continued to decrease. Few cells were isolated from the marrow cavities and spleens. Histology revealed fibrosis of the marrow and spleen and significant osteosclerosis of the marrow. An extramedullary hematopoiesis was observed in numerous organs such as the liver or the kidney. Total numbers of progenitors were very low in hematopoietic organs. Mice died 7 months after transplantation with severe pancytopenia. Two early deaths were observed with a marked increase in blast cells. This disorder was transplantable into secondary recipients who developed an attenuated form of the disease similar to the one previously described [Yan et al (1995) Blood 86: 4025]. In conclusion, dysregulated TPO production by hemopoietic cells in mice results in a fatal myeloproliferative disease which mimics the clinical evolution of idiopathic myelofibrosis observed in man.     

Allogeneic bone marrow transplantation in aggressive non-Hodgkin's lymphoma (excluding Burkitt and lymphoblastic lymphoma): a series of 73 patients from the SFGM database

The place of allogeneic bone marrow transplantation (BMT) in the treatment of aggressive non-Hodgkin's lymphoma (NHL) remains controversial. We conducted a retrospective study of French experience in allografting NHL between 1984 and 1994. To improve the homogeneity of the study population, cases of low-grade, Burkitt and lymphoblastic NHL were excluded. 73 patients were included in the analysis. Median age at transplantation was 35 years (range 9-61 years); 64 patients were in stage IV and 45 had bone marrow involvement at diagnosis. At the time of transplantation, 46 patients had sensitive disease (25 in complete remission; CR). The overall survival (OS) and progression-free survival (PFS) rates were 41% and 40% respectively at 5 years (median follow-up of survivors 90 months). The probability of disease progression was 30% at 5 years, and only one relapse occurred after 15 months. 32 patients died of transplantation-related complications. In multivariate analysis, pretransplant complete remission was the main factor associated with longer survival (OS at 60 months of 76% among the 25 patients in CR at transplant and of 23% among the 48 patients not in CR at transplant). Neither acute nor chronic graft-versus-host disease (GvHD) influenced the relapse rate. In conclusion, in this high-risk population the overall results of allogeneic BMT were encouraging, despite a high transplant-related mortality rate. We believe this procedure should be studied further in prospective controlled trials.