Among 94 osteopetrotic patients presenting with a severe clinical picture and diagnosed early in life, 12 bore mutations in the ClCN7 gene, but only 7 of them had the expected two recessive mutations. The remaining five patients seem to be heterozygous for a ClCN7 mutation, and significant variations were observed in the clinical manifestations of their disease, even within the same family.Introduction: Human osteopetroses are a heterogeneous group of diseases that include both infantile severe, autosomal recessive (ARO) and adult autosomal dominant (ADO) forms. Two genes, Atp6a3 (TCIRG1) and ClCN7, have been shown to be associated with human ARO, the latter of which is also thought to be responsible for ADO-II. However, patients with an intermediate phenotype have been described: the genetic basis of these observances is unknown. Materials and Methods:In this study, we report the clinical and molecular analysis of 94 patients in which a diagnosis of severe osteopetrosis was made within the first 2 years of age. Both TCIRG1 and CLCN7 genes were sequenced in all patients and the molecular findings were correlated to clinical parameters. Results and Conclusions:In 56 of 94 patients with a classical picture of ARO, TCIRG1-dependent recessive mutations were found. In contrast, ClCN7 mutations were found in 12 cases (13%) of severe osteopetrosis, but only 7 of them had two recessive mutations identified: in 6 of these 7 cases, central nervous system manifestations were noted, and these patients had a poor prognosis. The remaining five cases were heterozygous for a ClCN7 mutation,

T oxoplasma gondii is a protozoan parasite distributed worldwide and infecting one-third of the global population. Infection is acquired by ingestion of parasites via consumption of undercooked meat containing tissue cysts or of water, fruits, or vegetables contaminated with oocysts. In immunocompetent individuals, primary infection results in the formation of tissue cysts and in serological evidence of infection. However, in immunocompromized patients, such as hematopoietic stem cell transplant (HSCT) recipients, toxoplasmosis is often a life-threatening opportunistic infection arising either from transmission of the parasites via a graft from a seropositive donor to a seronegative recipient or, far more frequently, from reactivation of a preexisting latent infection in a seropositive recipient, regardless of the donor's serological status (1). After allogeneic HSCT, studies have shown an incidence of invasive toxoplasmosis among seropositive recipients of 4% to 6%, with an estimated mortality rate of 60% to 90% (2, 3).Nijmegen breakage syndrome (NBS) is a rare, autosome-recessive DNA repair disorder characterized by microcephaly with normal intelligence, facial dysmorphia (bird-like facial features), primary immunodeficiency, and a predisposition to lymphoid malignancies at a young age (4, 5). Treatment of NBS-related malignancies is challenging due to the chromosomal instability and immunodeficiency which make the patients more susceptible to the toxic effects of standard chemotherapy and radiation. Although lymphoid malignancies occurring in NBS patients can be successfully brought into remission using standard chemotherapy regimens with minor dose modifications (6, 7), a high rate of treatment failure and relapse has been observed (8). An alternative treatment option for malignancies in NBS is HSCT from HLAidentical donors, which has been shown not only to correct humoral and cellular immunodeficiency but also to lower the secondary malignancy rate (9).We here present a case of an early and fulminant post-HSCT reactivation of toxoplasmosis caused by an atypical T. gondii strain in a young patient with NBS that contributed to a subsequent fatal outcome within 40 days after transplantation. This case is the first report of increased clinical severity of toxoplasmosis caused by an atypical strain in the setting of immunosuppression. It also stresses the value and necessity of the use of quantitative molecular methods to monitor T. gondii reactivation in cases of reactivation risk.(The results of this study have been presented in part at the 11th European Multicolloquium of Parasitology [EMOP XI],

BackgroundFanconi anemia (FA) is characterized by sensitivity to DNA cross-linking agents, mild cellular, and marked clinical radio sensitivity. In this study we investigated telomeric abnormalities of non-immortalized primary cells (lymphocytes and fibroblasts) derived from FA patients of the FA-D2 complementation group, which provides a more accurate physiological assessment than is possible with transformed cells or animal models.ResultsWe analyzed telomere length, telomere dysfunction-induced foci (TIFs), sister chromatid exchanges (SCE), telomere sister chromatid exchanges (T-SCE), apoptosis and expression of shelterin components TRF1 and TRF2. FANCD2 lymphocytes exhibited multiple types of telomeric abnormalities, including premature telomere shortening, increase in telomeric recombination and aberrant telomeric structures ranging from fragile to long-string extended telomeres. The baseline incidence of SCE in FANCD2 lymphocytes was reduced when compared to control, but in response to diepoxybutane (DEB) the 2-fold higher rate of SCE was observed. In contrast, control lymphocytes showed decreased SCE incidence in response to DEB treatment. FANCD2 fibroblasts revealed a high percentage of TIFs, decreased expression of TRF1 and invariable expression of TRF2. The percentage of TIFs inversely correlated with telomere length, emphasizing that telomere shortening is the major reason for the loss of telomere capping function. Upon irradiation, a significant decrease of TIFs was observed at all recovery times. Surprisingly, a considerable percentage of TIF positive cells disappeared at the same time when incidence of γ-H2AX foci was maximal. Both FANCD2 leucocytes and fibroblasts appeared to die spontaneously at higher rate than control. This trend was more evident upon irradiation; the percentage of leucocytes underwent apoptosis was 2.59- fold higher than that in control, while fibroblasts exhibited a 2- h delay before entering apoptosis.ConclusionThe results of our study showed that primary cells originating from FA-D2 patients display shorten telomeres, elevated incidence of T-SCEs and high frequency of TIFs. Disappearance of TIFs in early response to irradiation represent distinctive feature of FANCD2 cells that should be examined further.

Deletions within chromosome 11q22-23, are considered among the most common chromosomal aberrations in chronic lymphocytic leukemia (CLL), and are associated with a poor outcome. In addition to the ataxia telangiectasia mutated (ATM) gene, the baculoviral IAP repeat-containing 3 (BIRC3) gene is also located in the region. BIRC3 encodes a negative regulator of the non-canonical nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) protein. Disruption of BIRC3 is known to be restricted to CLL fludarabine-refractory patients. The aim of the present study was to determine the frequency of copy number changes of BIRC3 and to assess its association with two known predictors of negative CLL outcome, ATM and tumor protein 53 (TP53) gene deletions. To evaluate the specificity of BIRC3 alterations to CLL, BIRC3 copy numbers were assessed in 117 CLL patients in addition to 45 B-cell acute lymphocytic leukemia (B-ALL) patients. A commercially available multiplex ligation dependent probe amplification kit, which includes four probes for the detection of TP53 and four probes for ATM gene region, was applied. Interphase-directed fluorescence in situ hybridization was used to apply commercially available probes for BIRC3, ATM and TP53. High resolution array-comparative genomic hybridization was conducted in selected cases. Genetic abnormalities of BIRC3 were detected in 23/117 (~20%) of CLL and 2/45 (~4%) of B-ALL cases. Overall, 20 patients with CLL and 1 with B-ALL possessed a BIRC3 deletion, whilst 3 patients with CLL and 1 with B-ALL harbored a BIRC3 duplication. All patients with an ATM deletion also carried a BIRC3 deletion. Only 2 CLL cases possessed deletions in BIRC3, ATM and TP53 simultaneously. Evidently, the deletion or duplication of BIRC3 may be observed rarely in B-ALL patients. BIRC3 duplication may occur in CLL patients, for which the prognosis requires additional studies in the future. The likelihood that TP53 deletions occur simultaneously with BIRC3 and/or ATM aberrations is low. However, as ATM deletions may, but not always, associate with BIRC3 deletions, each region should be considered in the future diagnostics of CLL in order to aid treatment decisions, notably whether to treat with or without fludarabine.

Severe combined immunodeficiency (SCID), including the 'variant' Omenn syndrome (OS), represent a heterogeneous group of monogenic disorders characterized by defect in differentiation of T- and/or B lymphocytes and susceptibility to infections since birth. In the period of 25 years, between January 1986 and December 2010, a total of 21 patients (15 SCID, 6 OS) were diagnosed in Mother & Child Health Institute of Serbia, a tertiary-care teaching University hospital and a national referral center for patients affected with primary immunodeficiency (PID). The diagnoses were based on anamnestic data, clinical findings, and immunological and genetic analysis. The median age at the onset of the first infection was the 2nd month of life. Seven (33 %) patients had positive family history for SCID. Out of five male infants with T-B+NK- SCID phenotype, mutation analysis revealed interleukin-2 (common) gamma-chain receptor (IL2RG) mutations in 3 with positive X-linked family history, and Janus-kinase (JAK)-3 gene defects in the other two. Six patients had T-B-NK+ SCID phenotype and further 6 features of OS, 11 of which had recombinase-activating gene (RAG1or RAG2) and 1 Artemis gene mutations. One child with T+B+NK+ SCID phenotype as well had proven RAG mutation. One child each with T-B+NK+ SCID phenotype, CD8 lymphopenia and unknown phenotype remained without known underlying genetic defect. Of the eight patients who underwent hematopoetic stem cell transplant (HSCT) 5 survived, the other 13 died between 2 days and 12 months after diagnosis was made. Early diagnosis of SCID, before onset of severe infections, offers possibility for HSCT and cure. Education of primary-care pediatricians, in particular including awareness of the risk of using live vaccines and non-irradiated blood products, should improve prognosis of SCID in our setting.

Survival of patients with high-risk pediatric solid tumors has improved with the introduction of a high-dose chemotherapy regimen and autologous stem cell rescue. Here, we present our data regarding the evaluation of the efficacy and safety of hematopoietic stem cell mobilization and harvesting in children with solid tumors. From November 2002 to March 2010, 85 children underwent autologous peripheral blood stem cell collection; 35 (41.1%) of them weighed less than 20 kg and were diagnosed with neuroblastoma, Wilms' tumor, medulloblastoma, yolk sac sarcoma, or non-Hodgkin's lymphoma. The mobilization regimens included disease-specific chemotherapy plus granulocyte colony-stimulating factor in most of the patients. The median age and weight at the time of apheresis was 36 months and 13.5 kg, respectively. Large-volume leukapheresis was performed with the aim of reducing the psychological and financial impact of leukapheresis by reducing the number of procedures while collecting a large number of cells. The median number of mobilization and leukapheresis procedures per case was one. The pre-apheresis CD34+ cell count ranged from 2 to 845 µL, with a median of 24 µL. A median of four patient blood volumes was processed per procedure, lasting 279 min (range, 113-420 min). A radial catheter was used for harvesting in 35 procedures (71.4%). The median yield of CD34+ cells was 6.6×10(6) /kg per patient. The targeted dose of 5×10(6) /kg CD34+ cells was realized in 80% of patients. The tolerance of peripheral blood stem cell collection in our patients was good. In conclusion, the collection of peripheral blood stem cells is an effective and safe procedure, even when conducted on the youngest children.

Objectives: Reactivation of latent toxoplasmosis may be life-threatening in haematopoietic stem cell transplant (HSCT) recipients. We conducted an 8-year-long prospective study on the diagnosis and monitoring of reactivated toxoplasmosis in paediatric HSCT recipients. The primary objective was to determine the incidence of reactivated toxoplasmosis in a setting that withholds prophylaxis until engraftment. The second objective was to identify the subgroups of HSCT recipients particularly prone to reactivation who may benefit the most from regular PCR follow-up. Methods: Serological and qPCR screening targeting the Toxoplasma 529 bp gene was performed before HSCT, and continued by weekly monitoring after HSCT for a median time of 104 days. Results: Reactivated toxoplasmosis was diagnosed in 21/104 (20.2%), predominantly in allo-(19/75) and rarely in auto-HSCT (2/29) recipients. Over 50% (14/21) of cases were diagnosed during the first month after HSCT, while awaiting engraftment without prophylaxis. Toxoplasma disease evolved in only three (14.3%, 3/21) patients, all treated by allo-HSCT. Reactivation was more frequent in patients treated for acute lymphoblastic leukaemia (3/27, p 0.03) and especially, in recipients of haploidentical stem cells (10/ 20, p 0.005). Seronegative status of the donor (where was known) contributed to 75% (12/16) cases of reactivated toxoplasmosis after allo-HSCT. Discussion: The presented results show that peripheral blood-based qPCR, both before and after HSCT, is a valuable asset for the diagnosis of reactivated toxoplasmosis, whereas the results of serology in recipients should be interpreted with caution. Weekly qPCR monitoring, at least until successful engraftment and administration of prophylaxis, allows for prompt introduction of specific treatment.

Fanconi anemia (FA) is a rare cancer-prone genetic disorder characterized by progressive bone marrow failure, chromosomal instability and redox abnormalities. There is much biochemical and genetic data, which strongly suggest that FA cells experience increased oxidative stress. The present study was designed to elucidate if differences in oxidant state exist between control, idiopathic bone marrow failure (idBMF) and FA cells, and to analyze oxidant state of cells in FA heterozygous carriers as well. The results of the present study confirm an in vivo prooxidant state of FA cells and clearly indicate that FA patients can be distinguished from idBMF patients based on the oxidant state of cells. Female carriers of FA mutation also exhibited hallmarks of an in vivo prooxidant state behaving in a similar manner as FA patients. On the other hand, the oxidant state of cells in FA male carriers and idBMF families failed to show any significant difference vs. controls. We demonstrate that the altered oxidant state influences susceptibility of cells to apoptosis in both FA patients and female carriers. The results highlight the need for further research of the possible role of mitochondrial inheritance in the pathogenesis of FA.