New αIIbß3 variants in 28 Turkish Glanzmann patients; structural hypothesis for complex activation by residues variations in I-EGF domains.

Glanzmann thrombasthenia (GT) is a rare autosomal recessive bleeding disorder characterized by impaired platelet aggregation due to defects in integrin αIIbß3, a fibrinogen receptor. Platelet phenotypes and allelic variations in 28 Turkish GT patients are reported. Platelets αIIbß3 expression was evaluated by flow cytometry. Sequence analyzes of ITGA2B and ITGB3 genes allowed identifying nine variants. Non-sense variation effect on αIIbß3 expression was studied by using transfected cell lines. 3D molecular dynamics (MDs) simulations allowed characterizing structural alterations. Five new alleles were described. αIIb:p.Gly423Asp, p.Asp560Ala and p.Tyr784Cys substitutions impaired αIIbß3 expression. The αIIb:p.Gly128Val substitution allowed normal expression; however, the corresponding NM_000419.3:c.476G>T variation would create a cryptic donor splicing site altering mRNA processing. The ß3:p.Gly540Asp substitution allowed αIIbß3 expression in HEK-293 cells but induced its constitutive activation likely by impairing αIIb and ß3 legs interaction. The substitution alters the ß3 I-EGF-3 domain flexibility as shown by MDs simulations. GT variations are mostly unique although the NM_000419.3:c.1752 + 2 T > C and NM_000212.2:c.1697 G > A variations identified in 4 and 8 families, respectively, might be a current cause of GT in Turkey. MD simulations suggested how some subtle structural variations in the ß3 I-EGF domains might induce constitutive activation of αIIbß3 without altering the global domain structure.

Clinical manifestations of infants with nutritional vitamin B deficiency due to maternal dietary deficiency.

AIM: In developing countries, nutritional vitamin B(12) deficiency in infants due to maternal diet without adequate protein of animal origin has some characteristic clinical features. In this study, haematological, neurological and gastrointestinal characteristics of nutritional vitamin B(12) deficiency are presented. METHODS: Hospital records of 27 infants diagnosed in a paediatric haematology unit between 2000 and 2008 were evaluated retrospectively. RESULTS: The median age at diagnosis was 10.5 months (3-24 months). All the infants were exclusively breast fed and they presented with severe nonspecific manifestations, such as weakness, failure to thrive, refusal to wean, vomiting, developmental delay, irritability and tremor in addition to megaloblastic anaemia. The diagnosis was confirmed by complete blood counts, blood and marrow smears and serum vitamin B(12) and folic acid levels. The median haemoglobin level was 6.4 g/dL (3.1-10.6) and mean corpuscular volume (MCV) was 96.8 fL (73-112.3). Some patients also had thrombocytopaenia and neutropaenia. All the infants showed clinical and haematological improvement with vitamin B(12) administration. Patients with severe anaemia causing heart failure received packed red blood cell transfusions as the initial therapy. CONCLUSION: Paediatricians must consider nutritional vitamin B(12) deficiency due to maternal dietary deficiency in the differential diagnosis of some gastrointestinal, haematological, developmental and neurological disorders of infants with poor socioeconomic status. Delay in diagnosis may cause irreversible neurological damage.

A rare syndrome in the differential diagnosis of hepatosplenomegaly and pancytopenia: report of identical twins with Griscelli disease.

White, identical twin boys aged 3 months were referred to our centre with persisting fever, mouth ulcers, hepatosplenomegaly, pancytopenia and failure to thrive. The parents were first cousins and there was a history of a sibling with similar manifestations who had died. The infants had silvery-grey hair and pigment clumps on the hair shafts, and skin biopsy showed accumulation of melanocytes on melanosomes. Bone marrow revealed hypercellularity and haemophagocytosis. HLH-94 chemotherapy (initial therapy with daily dexamethasone and etoposide, maintenance with dexamethasone pulses, etoposide and cyclosporin A) was started. Though partial haematological remission was achieved, one of the boys died on the 34th day following aspiration pneumonia. No pathogen could be identified. The second boy responded to therapy but had a haematological relapse and died 68 days after first being admitted. Genetic study revealed a 5 bp deletion in the RAB27A gene (510 del AAGCC in exon 5). Transient haematological remission can be achieved with chemotherapy but allogeneic bone marrow transplantation is the only curative therapy in Griscelli disease, as in other familial haemophagocytic syndromes. Identification of the mutation also provides an opportunity for prenatal diagnosis.

Fludarabine, cytarabine, G-CSF and idarubicin (FLAG-IDA) for the treatment of relapsed or poor risk childhood acute leukemia.

The prognosis of relapsed acute leukemia or chronic leukemia in acute blast crisis is poor and new chemotherapeutic regimens could be useful for these patients. Six relapsed acute lymphoblastic leukemia (ALL), nine relapsed acute myeloblastic leukemia (AML), one chronic myelomonocytic leukemia (CMML) and one chronic myeloid leukemia (CML) in acute blast crisis between three to 18 years (median 10 years) received fludarabine, cytarabine, G-CSF and idarubicin (FLAG-IDA) chemotherapy (CT). Five of the AML relapses were after bone marrow transplantation (BMT) and four were recurrent relapses. At the end of the second course only three patients (2 AML, 1 ALL) were in complete remission (CR). Of the three patients in CR, one patient with AML had her first donor lymphocyte transfusion (DLT) on the 7th day of the second FLAG-IDA course and she is disease-free on the 30th month of the second remission. The remaining two patients were transplanted from unrelated donors in a BMT center abroad on the 5th and 8th month of the last remission and both died with BMT-related complications. Out of 25 courses, seven resulted in fatal infections. The regimen was ineffective in B-cell ALL as in acute blastic crisis of CMML and CML. We could not evaluate the remission-inducing effect accurately in most of the patients due to induction failure. FLAG-IDA appears to be a myelotoxic therapy for relapsed or poor risk leukemia in a developing country. It is not cost-effective; dose modifications or a regimen without IDA may be tried if there is an available marrow donor.

Is AML1/ETO gene expression a good prognostic factor in pediatric acute myeloblastic leukemia?

To assess the clinical significance of AML1/ETO gene detected by nested reverse transcriptase polymerase chain reaction, the outcome of 7 patients with acute myeloblastic leukemia between 3 and 14 years of age were presented. All patients had complete remission (CR) at the end of induction (AML-MRC 10 protocol) and 4 underwent unpurged autologous, 2 allogeneic (from matched siblings) non-T-cell-depleted bone marrow transplantations (BMT) in first CR. One patient died due to allogeneic BMT-related complications, and 4 patients relapsed at 13, 17, 18, and 26 months. Only one patient achieved second CR. All relapsed patients died between 18 and 36 months with resistant disease (n = 3) or infection during salvage chemotherapy (n = 1). Two patients who had autologous BMT are alive and disease free at 44 and 50 months. Although statistical significance could not be shown, event-free survival and overall survival rates of AML1/ETO-positive patients (28.57 and 28.57%, respectively) at 3.5 years were even lower than those of AML1/ETO-negative patients. The results confirm some previous reports that AML1/ETO gene in children and adolescents is not a favorable prognostic factor.

Could Parvovirus B19 Induce a Rejection After Bone Marrow Transplantation in a Patient with Diamond-Blackfan Anemia?

A four year-old-girl with Diamond-Blackfan anemia (DBA) that was resistant to corticosteroid treatment and transfusion dependent underwent (bone marrow transplantation) BMT from HLA identical sibling. The patient was conditioned with busulfan and cyclophosphamide and achieved complete marrow engraftment and mixed chimerism in DNA analysis. For the following 13 months she was not transfusion dependent and had a 100% Karnofsky score. But on the 14th month she had anemia ollowing fever, rash and enteritis. Parvovirus B19 IgM seropositivity confirmed Parvovirus infection. Although intravenous immunoglobulin was administered, bone marrow morphology and DNA analysis revealed rejection. Although mixed chimerism detected shortly after the BMT procedure might raise the possibility of an ongoing slow graft rejection during the relatively stable remission period, we think that parvovirus B19 had also contributed rejection.

Prognostic significance of wilms tumor 1 gene in childhood acute Lymphoblastic Leukemia.

Wilms tumor 1 (WT1) gene is a tumor supressor gene, expressed in malignant and normal hematopoietic progenitor cells. Prognostic significance of this gene in childhood acute lymphoid leukemia (ALL) is not clear. We evaluated the presence of WT1 expression in bone marrow samples of 28 children with de novo ALL at diagnosis by two step RT-PCR. Expression of WT1 gene was detected in 78.5% of patients. There was no correlation between WT1 gene expression and age, sex, FAB type, leukocyte count, and presence of t(4;11) and t(9;22). All patients were treated with modified BFM 86 protocol. There was no difference in the complete remission (CR) rate between WT1 positive and negative patients. Event free survival (EFS) and overall survival (OS) of WT1 positive and negative patients were also not significant. We conclude that expression of WT1 gene is not associated with specific characteristics of ALL blast cells and is not a prognostic factor for CR, remission duration and overall survival.

Detection of BCR/ABL transcripts by reverse transcriptase polimerase chain reaction in pediatric acute Lymphoblastic Leukemia: incidence and clinical eatures.

BCR/ABL expression, which is the molecular equivalent of the Philadelphia chromosome, is an independent poor risk factor in acute lymphoblastic leukemia (ALL). We used a two-step (nested) reverse transcriptase polimerase chain reaction (RT-PCR) assay to examine BCR/ABL expression in the diagnostic bone marrow specimen of children with ALL, prospectively. Among 75 de novo ALL patients, 4 (%5.3) were found to be BCR/ABL- ositive, whereas 4 of 17 relapsed patients (23.5%) were positive. This preliminary study in Turkish children showed an incidence similar to reports from Europe and the U.S.A. More intensive chemotherapies and allogeneic bone marrow transplantations (BMT) uring the first remission were planned if a donor was available. Out of 8 BCR/ABL-positive patients, complete remission (CR) was achieved in 7 patients and partial remission (PR) was achieved in 1 patient. Three patients underwent allogeneic BMT during the first CR and 1 under went autologous BMT during the first PR. The Kaplan-Meier estimate of vent-free survival (EFS) of BCR/ABL negative de novo ALL patients was 78.36% at 3 years, whereas the EFS of positive patients was 31.25% at 26 ± 6.4 months. Molecular screening for the Philadelphia chromosome should become a part of the routine diagnostic panel in ALL patients in order to predict which patients have a poor prognosis and need tailored therapy.