Mualla Cetin

NBEAL2 is mutated in gray platelet syndrome and is required for biogenesis of platelet α-granules

Gray platelet syndrome (GPS) is an autosomal recessive bleeding disorder that is characterized by large platelets that lack α-granules. Here we show that mutations in NBEAL2 (neurobeachin-like 2), which encodes a BEACH/ARM/WD40 domain protein, cause GPS and that megakaryocytes and platelets from individuals with GPS express a unique combination of NBEAL2 transcripts. Proteomic analysis of sucrose-gradient subcellular fractions of platelets indicated that NBEAL2 localizes to the dense tubular system (endoplasmic reticulum) in platelets.

LAD-1/variant syndrome is caused by mutations in FERMT3

Leukocyte adhesion deficiency-1/variant (LAD1v) syndrome presents early in life and manifests by infections without pus formation in the presence of a leukocytosis combined with a Glanzmann-type bleeding disorder, resulting from a hematopoietic defect in integrin activation. In 7 consanguineous families, we previously established that this defect was not the result of defective Rap1 activation, as proposed by other investigators. In search of the genetic defect, we carried out homozygosity mapping in 3 of these patients, and a 13-Mb region on chromosome 11 was identified. All 7 LAD1v families share the same haplotype, in which 3 disease-associated sequence variants were identified: a putative splice site mutation in CALDAGGEF1 (encoding an exchange factor for Rap1), an intronic 1.8-kb deletion in NRXN2, and a premature stop codon (p.Arg509X) in FERMT3. Two other LAD1v patients were found to carry different stop codons in FERMT3 (p.Arg573X and p.Trp229X) and lacked the CALDAGGEF1 and NRXN2 mutations, providing convincing evidence that FERMT3 is the gene responsible for LAD1v. FERMT3 encodes kindlin-3 in hematopoietic cells, a protein present together with integrins in focal adhesions. Kindlin-3 protein expression was undetectable in the leukocytes and platelets of all patients tested. These results indicate that the LAD1v syndrome is caused by truncating mutations in FERMT3.

Gray platelet syndrome: natural history of a large patient cohort and locus assignment to chromosome 3p

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. Reports on GPS are limited to case presentations. The causative gene and underlying pathophysiology are largely unknown. We present the results of molecular genetic analysis of 116 individuals including 25 GPS patients from 14 independent families as well as novel clinical data on the natural history of the disease. The mode of inheritance was autosomal recessive (AR) in 11 and indeterminate in 3 families. Using genome-wide linkage analysis, we mapped the AR-GPS gene to a 9.4-Mb interval on 3p21.1-3p22.1, containing 197 protein-coding genes. Sequencing of 1423 (69%) of the 2075 exons in the interval did not identify the GPS gene. Long-term follow-up data demonstrated the progressive nature of the thrombocytopenia and myelofibrosis of GPS resulting in fatal hemorrhages in some patients. We identified high serum vitamin B(12) as a consistent, novel finding in GPS. Chromosome 3p21.1-3p22.1 has not been previously linked to a platelet disorder; identification of the GPS gene will likely lead to the discovery of novel components of platelet organelle biogenesis. This study is registered at www.clinicaltrials.gov as NCT00069680 and NCT00369421.

Life-threatening neurological complications after bone marrow transplantation in children

Summary:Neurological complications may occur in BMT recipients (11–59%), frequently contributing to morbidity or mortality. They are the main causes of death in 10–15%. Life-threatening neurological complications were seen in 11 out of 113 (9.7%) children who underwent BMT from HLA-matched family (n=7) or mismatched donors (n=4) at our institution. Diagnoses of patients with neurological complications were acute myeloblastic leukemia (AML) (five), thalassemia major (two), Fanconi anemia (two), Omenn syndrome (one) and leukodystrophy (one), and the neurological events were seen between days +13 and +85 after transplantation. Minor symptoms including reversible, nonrepetitive seizures were excluded. Cyclosporine A toxicity was diagnosed in six children. The rest of the complications were brain abscess/meningoencephalitis (two), severe hypomagnesemia (one), busulfan toxicity (one), sustained hypertension (three), and intracranial hemorrhage (three). Six patients with neurological complications suffered from >grade II graft-versus-host disease (GvHD), and all were high risk for transplant-related complications. In this study, risk status of the underlying disease, mismatched transplantation, a diagnosis of AML (advanced stage), older age and >grade II GvHD were important adverse factors for the development of severe life-threatening neurological complications.

Secondary hemophagocytic lymphohistiocytosis in Turkish children.

Between January 1998 and January 2005, a total of 18 children 2 weeks–72 months of age were diagnosed as having secondary hemophagocytic lymphohistiocytosis. The frequency of secondary hemophagocytic lymphohistiocytosis among total hospitalized patients during this period was 0.05% (18 of 34,250). Of the 18 patients, 8 (44.5%) had bacterial infections; cytomegalovirus and Epstein-Barr virus infections were present in 5 (28%) and 1 (5.5%), patient, respectively. Leishmaniasis was diagnosed in 2 patients (11%), and herpes simplex virus was diagnosed in 2 patients (11%). Six patients died during treatment, and 1 patient was lost to follow-up. The survival rate was 61%.

Primary hemophagocytic lymphohistiocytosis in Turkish children

Nineteen children with hemophagocytic lymphohistiocytosis (HLH) were studied in the Department of Pediatric Hematology, Hacettepe University. Patients were divided into two groups. Group 1 : Thirteen patients were classified as having a genetic etiology (7 familial, 6 presumed familial) on the basis of an affected sibling and consanguinity. There was a history of consanguineous marriage in 13 of the families. Seven of them had a history of a sibling with HLH. Group 2 : Six patients were diagnosed with sporadic HLH. The age at presentation for familial patients was 0.7-84 months (mean 21.9 - 24.9 months), and for sporadic cases it was 2.5-48 months (mean 22.7 - 19.8 months). The clinical and laboratory data of these two groups were similar at diagnosis. Thirteen cases were diagnosed premortem by bone marrow aspiration. Splenic biopsy was performed in 2 patients. Four patients were diagnosed by postmortem examination. Elevated LDH levels were found in all patients tested. No significant differences for clinical and laboratory data were found between the two groups.

Benefit of high-dose methylprednisolone in comparison with conventional-dose prednisolone during remission induction therapy in childhood acute lymphoblastic leukemia for long-term follow-up.

Eight-year event-free survival (EFS) was evaluated in 205 patients with acute lymphoblastic leukemia (ALL), to consider the efficacy of high-dose methylprednisolone (HDMP) given during remission induction chemotherapy between 1 and 29 days. The St Jude Total XI Study protocol was used after some minor modifications in this trial. Patients were randomized into two groups. Group A (n = 108) received conventional dose (60 mg/m2/day orally) prednisolone and group B (n = 97) received HDMP (Prednol-L, 900–600 mg/m2 orally) during remission induction chemotherapy. Complete remission was obtained in 95% of the 205 patients who were followed-up for 11 years; median follow-up was 72 months (range 60–129) and 8-year EFS rate was 60% overall (53% in group A, 66% in group B). The EFS rate of group B was significantly higher than of group A (P = 0.05). The 8-year EFS rate of groups A and B in the high-risk groups was 39% vs 63% (P = 0.002). When we compared 8-year EFS rate in groups A and B in the high-risk subgroup for both ages together ⩽2 or ⩾10 years, it was 44% vs 74%, respectively. Among patients in the high-risk subgroup with a WBC count ⩾50 × 109/l, the 8-year EFS was 38% in group A vs58% in group B. During the 11-year follow-up period, a total of 64 relapses occurred in 205 patients. In group A relapses were higher (39%) than in group B (23%) (P = 0.05). These results suggest that HDMP during remission-induction chemotherapy improves the EFS rate significantly for high-risk patients in terms of the chances of cure.

Evaluation of children with myelodysplastic syndrome: importance of extramedullary disease as a presenting symptom.

Thirty-three children diagnosed with primary myelodysplastic syndrome (MDS) in a single institution over an 8 year period were evaluated with special emphasis on children who presented with extramedullary disease (EMD). EMD was present at diagnosis in 12 (36%) of the 33 children with MDS. Three patients with juvenile myelomonocytic leukemia (JMML) and 2 patients with chronic myelomonocytic leukemia (CMML) presented with pleural effusion. Pericardial effusion was present in 3 of these patients, two of whom also had thrombosis. Pyoderma gangrenosum, relapsing polychondritis were the initial findings in another two cases with JMML. Lymphadenopathy (n=1), gingival hypertrophy (n=2), orbital granulocytic sarcoma (n=1) and spinal mass (n=1) were the presenting findings in 5 patients with refractory anemia with excess of blasts in transformation. Since high-dose methylprednisolone (HDMP, 20–30 mg/kg/day) has been shown to induce differentiation and apoptosis of myeloid leukemic cells in children with different morphological subtypes of acute myeloid leukemia in vivo and in vitro, 25 children with de novo MDS were treated with combined HDMP and cytotoxic chemotherapy. Dramatic improvement of EMD and decrease in blast cells both in the peripheral blood and bone marrow were obtained following administration of short-course HDMP treatment alone as observed in children with AML. HDMP, combined with low-dose cytosine arabinoside and mitoxantrone were used for the remission induction. Remission was achieved in 8 (80%) of 10 children who presented with EMD and in 9 (60%) of 15 children without EMD. Long-term remission (>6 years) was obtained in 4 (two with JMML and two with CMML), three of whom presented with EMD. In conclusion EMD can be a presenting finding in childhood MDS as observed in adults. In addition, the beneficial effect of HDMP combined with more intensive chemotherapy should be explored as alternative therapy in children with MDS not suitable for bone marrow transplantation.

Alternative algorithm for L-asparaginase allergy in children with acute lymphoblastic leukemia

BACKGROUND L-asparaginase is a crucial chemotherapeutic agent for the treatment of acute lymphoblastic leukemia. The alternatives to L-asparaginase are not available in many parts of the world, including Turkey. OBJECTIVE We sought to evaluate the utility of premedication with or without a desensitization protocol in children with acute lymphoblastic leukemia and systemic hypersensitivity reactions to Escherichia coli-asparaginase. METHODS In this prospective study patients with systemic hypersensitivity reactions to E coli-asparaginase for whom we were unable to ascertain/provide other alternatives to asparaginase were either premedicated, desensitized, or both to receive their chemotherapy as E coli-asparaginase according to the severity of the hypersensitivity reaction. RESULTS Nineteen patients (13 male patients) with a mean age of 7.4 +/- 4.7 years experienced a systemic hypersensitivity reaction to E coli-asparaginase during a 4-year period. Polyethylene glycol-asparaginase could be used for 3 patients. Eight of the remaining 16 children, who had experienced anaphylaxis, were premedicated and desensitized with E coli-asparaginase, and in 7 patients treatment was tolerated. The other 8 patients, with acute allergic reactions to E coli-asparaginase, were premedicated first, and 5 of them showed no reaction subsequently. Three of them demonstrated systemic hypersensitivity reactions again (anaphylaxis, n = 3), and premedication and desensitization with E coli-asparaginase resulted in anaphylaxis. Polyethylene glycol-asparaginase was administered uneventfully to the patients who could be provided it. CONCLUSION E coli-asparaginase could be administered to more than half of the patients who had a hypersensitivity reaction, and all of these patients were able to receive their planned doses of asparaginase. In countries with shortages of alternative asparaginase preparations, our approach might be a suitable option.

Morphologic Evidence of Apoptosis in Childhood Acute Myeloblastic Leukemia Treated with High-Dose Methylprednisolone

We have previously demonstrated that various subtypes of AML children respond to high-dose methylprednisolone (HDMP; 20-30 mg/kg/day) which could induce in vivo differentiation of myeloid leukemic cells to mature granulocytes. In this study we have evaluated whether apoptosis occurs in AML cells of patients treated by HDMP using morphological criteria. For light and electron microscopic examination bone marrow aspirates were obtained four days and two weeks after methylprednisolone (30 mg/kg/day) treatment from two children with newly diagnosed AML (AML-M3 and AML-M4). In both patients maturation of leukemic cells has previously been reported four days (in patient with AML-M3) and two weeks (in patient with AML-M4) after HDMP treatment. Electron microscopy revealed the characteristic ultrastructural changes of various stages of apoptosis four days after HDMP treatment in a case with AML-M3. Morphologic evidence of apoptosis induced by HDMP were also detected on Wright-stained and toluidine blue stained semithin sections of BM preparations in a patient with AML-M4 and AML-M3 respectively. These findings suggest that HDMP which could induce in vivo terminal differentiation in myeloid leukemic cells is also able to induce apoptosis in patients with AML. The possibility of HDMP-induced apoptosis should be evaluated in a larger series of patients with AML and other types of malignant tumors.