Zühre Kaya

Subtle differences in CTL cytotoxicity determine susceptibility to hemophagocytic lymphohistiocytosis in mice and humans with Chediak-Higashi syndrome.

Perforin-mediated cytotoxicity is important for controlling viral infections, but also for limiting immune reactions. Failure of this cytotoxic pathway leads to hemophagocytic lymphohistiocytosis (HLH), a life-threatening disorder of uncontrolled T-cell and macrophage activation. We studied susceptibility to HLH in 2 mouse strains (souris and beige(J)) and a cohort of patients with partial defects in perforin secretion resulting from different mutations in the LYST gene. Although both strains lacked NK-cell cytotoxicity, only souris mice developed all clinical and histopathologic signs of HLH after infection with lymphocytic choriomeningitis virus. The 2 strains showed subtle differences in CTL cytotoxicity in vitro that had a large impact on virus control in vivo. Whereas beige(J) CTLs eliminated lymphocytic choriomeningitis virus infection, souris CTLs failed to control the virus, which was associated with the development of HLH. In LYST-mutant patients with Chediak-Higashi syndrome, CTL cytotoxicity was reduced in patients with early-onset HLH, whereas it was retained in patients who later or never developed HLH. Thus, the risk of HLH development is set by a threshold that is determined by subtle differences in CTL cytotoxicity. Differences in the cytotoxic capacity of CTLs may be predictive for the risk of Chediak-Higashi syndrome patients to develop HLH.

Invasive fungal infections in pediatric leukemia patients receiving fluconazole prophylaxis.

Children with acute leukemia have increased risk for invasive fungal infections (IFI) but the role of long term antifungal prophylaxis (AFP) in morbidity and mortality of IFI is not well‐known.

Lung Function, Pulmonary Complications, and Mortality after Allogeneic Blood and Marrow Transplantation in Children

Pulmonary complications (PC) remain a significant barrier to the success of allogeneic blood and marrow transplantation (BMT). Pretransplant pulmonary function tests (PFTs) have been correlated with risk of early respiratory failure and mortality in adult BMT recipients. There is limited data on their relationship to posttransplant outcomes in pediatric patients. We sought, in pediatric allo-BMT recipients (1) to analyze the spectrum of infectious and noninfectious PCs, (2) to evaluate the prevalence and course of PFT abnormalities before and after transplant, and (3) to correlate pretransplant PFT findings with patient outcomes, specifically risk of PC, respiratory failure, and death. We conducted a retrospective review of PC in all patients who underwent allo-BMT at Childrens Hospital of Pittsburgh during 1996 to 2006. PFTs were performed in children 6 years and older pretransplant, 3, 6, 12, and 24 months after transplant. PCs occurring within 100 days of BMT were considered early. One hundred ten consecutive children who underwent allo-BMT were included (median age = 9.7 years; 67 males, 43 females). Seventy-five of 110 patients had 370 PFT studies performed; 62 of 73 patients >6 years of age (85%) underwent PFT studies pre-BMT. There was a significantly higher risk of early respiratory failure in patients with reduced pretransplant forced expiratory volume in 1 second (FEV(1)) (P = .0001, odds ratio [OR] 5.1) or forced vital capacity (FVC) (P = .0001, OR 8.5). Forty-three of 110 (39%) patients required mechanical ventilation, and in 30 episodes (70%), patients remained ventilator-dependent until time of death. Posttransplant, we observed statistically significant reductions in FEV(1), FVC, total lung capacity (TLC), and diffusing capacity of the lungs (DLco) at 3 months post-BMT and similar reductions at 6 months post-BMT except for DLco (not significant). Between 12 and 24 months, FEV(1), FVC, TLC, and DLco improved significantly from earlier declines post-BMT; however FEV(1) and FVC remained significantly below pretransplant values. At a median follow-up of 5.5 (1.6-11.6) years, 58 of 110 (53%) patients were surviving. The majority of the patients who died from transplant-related complications suffered from 1 or more PCs (31/32, 97%). Early PC was associated with over 4-fold reduction in probability of survival at 10 years (8/44, 18% with early PC versus 50/66, 76% without early PC). On multivariate analysis, risk of death was significantly associated with high-risk disease status (P = .015; hazard ratio [HR] = 2.5), unrelated donor (P = .03; HR = 2.1), early PC (P = .0001; HR = 7.7) and pathogen identification (P = .02; HR = 2.7). These results suggest that, in children undergoing allo-BMT (1) compromised pretransplant lung function is significantly correlated with risk of early respiratory failure but not of overall survival (OS), (2) reductions in lung volumes and diffusion capacity are common 3- to 6-month posttransplant with partial recovery by 12 to 24 months, (3) there is high mortality following mechanical ventilation, and (4) early PCs are associated with significantly worse OS.

A novel single point mutation of the LYST gene in two siblings with different phenotypic features of Chediak Higashi syndrome.

Chediak Higashi syndrome (CHS) is an autosomal‐recessive disorder characterized by oculocutaneous albinism, recurrent infections and a progressive primary neurological disease. Here, we describe two siblings with CHS due to a novel homozygous R1836X mutation in the LYST gene associated with loss of NK cell degranulation and cytotoxicity. While one sibling was born with fair skin and hair and died of hemophagocytic lymphohistiocytosis (HLH) at 5 months of age, the other sibling had dark black hair and skin and developed HLH at the age of 4 years. Pediatr Blood Cancer 2011;56:1136–1139.

Phenotypic heterogeneity and evidence of a founder effect associated with G6PC3 mutations in patients with severe congenital neutropenia

Causative homozygous or compound heterozygous mutations in the glucose-6-phosphatase, catalytic subunit 3 gene (G6PC3) have recently been described for a sub-group of patients with severe congenital neutropenia (SCN) (Arostegui et al, 2009; Boztug et al, 2009, 2011; Xia et al, 2009; Germeshausen et al, 2010; Banka et al, 2011a,b; Hayee et al, 2011). In addition to neutropenia and recurrent infections, mutant-positive patients are reported to have varied other congenital abnormalities, such as a prominent superficial venous pattern, structural heart defects, urogenital malformations, skeletal abnormalities, primary pulmonary hypertension, growth and developmental delay (Boztug et al, 2011). Several reports have also indicated considerable variation in bone marrow (BM) morphology, including a lack of the myeloid maturation arrest characteristic of SCN (McDermott et al, 2010; Banka et al, 2011b). We screened 108 CN patients or kindreds, known wild-type for the ELANE, HAX1 and WAS genes, for G6PC3 mutations and identified further phenotypic heterogeneity, in that two mutant-positive patients had neutropenia alone. Another presented with isolated neutropenia and was found to have clinically asymptomatic cardiac abnormalities. The study had Local Regional Ethical Committee approval. Informed consent was obtained from patients and/or their parents. Polymerase chain reaction (PCR) amplicons of the six G6PC3 exons were screened using denaturing high performance liquid chromatography (Transgenomic Limited, Glasgow, UK) (see Table S1). Samples with abnormal chromatograms were sequenced. Mutations were confirmed by PCR with mismatch primers and restriction enzyme digestion (Table S2). Three different homozygous G6PC3 mutations were detected in four of the 108 patients/kindreds studied. Parents of affected individuals were all heterozygous for the mutation. Patient 1 (born 1982, from Pakistan, parents first cousins) had a homozygous 21 bp deletion in exon 1 (c.190_210del) resulting in an in-frame deletion of seven amino acids in the second trans-membrane domain (p.Thr64_Ile70del) (Fig 1A, B). The patients’ phenotype was similar to that of the G6PC3-mutated patients described by Boztug et al (2009). In addition to neutropenia (absolute neutrophil count [ANC] 0·1 × 109/l), first noted aged 9 years, he suffered from recurrent oral ulcers, aspergillus infection and recurrent episodic bacterial pneumonia, but responded to standard antibiotic therapy and now receives regular granulocyte colony-stimulating factor (G-CSF). He was born with an atrial septal defect and has granulomatous inflammatory bowel disease, splenomegaly, digital clubbing and short stature. His affected sister is also homozygous G6PC3 mutant and has a similar phenotype but without the atrial septal defect and opportunistic infection. Fig 1 WAVE chromatograms, restriction enzyme digests and haplotype study of G6PC3 mutant-positive kindreds. (A) WAVE chromatograms of exon 1 and exon 5 polymerase chain reaction (PCR) products from mutation-positive patients mixed with known wild-type (WT) ... Patient 2 (born 2001, from Turkey, consanguineous parents) has a novel c.623T>G mutation in exon 5 resulting in p.Leu208Arg (Fig 1A, B). She presented in infancy with severe neutropenia (ANC <0·1 × 109/l), recurrent otitis media, chronic gingivitis and periodontitis. Initially responsive to 5 μg/kg G-CSF, subsequent doses in excess of 10–15 μg/kg have failed to control her infections. Although otherwise clinically asymptomatic and with no obvious congenital anomalies, echocardiography post-G6PC3 mutation detection showed a patent foramen ovale and tricuspid insufficiency. Patients 3 and 4, both from Pakistan, are unrelated but have the same homozygous missense mutation in exon 1, c.130C>T resulting in p.Pro44Ser (Fig 1A, B). Both have normal echocardiograms, lack other congenital abnormalities and have had normal growth and development. Patient 3 (born 1989, non-consanguineous parents) presented with neutropenia at age 13 years with intermittent mouth ulcers (ANC 0·4 × 109/l) and has suffered a single bout of myositis. His only treatment has been intermittent G-CSF for recurring severe mouth ulcers. Patient 4 (born 2002, parents first cousins) presented at 3 years of age with recurrent upper respiratory and chest infections, and has had two instances of gluteal abscesses. He has otherwise been well, with a recent ANC of 0·45 × 109/l, and has not received G-CSF. Of note, both patients have normocellular BM morphology with normal neutrophils, although slightly left-shifted myelopoiesis was reported for Patient 3. The mutation was not detected in samples from 54 haematologically normal individuals of Pakistani origin but was reported recently in an SCN patient, also of Pakistani origin, who similarly had normal BM cellularity without maturation arrest, although further information on the patient was not available (Banka et al, 2011b). Pro44 and Leu208 are both conserved amongst species (Fig S1) and, due to their trans-membranous location, the mutations are predicted to be disruptive to normal G6PC3 function. A possible ancestral founder for the p.Pro44Ser mutation was identified from a common haplotype consisting of two highly polymorphic micro-satellite markers and four single nucleotide polymorphisms spanning a maximum of 4·8Mb in the two unrelated p.Pro44Ser-mutated patients (Fig 1C) (PCR primers and conditions in Table S3). None of 20 Pakistani control samples carried the entire six-marker haplotype, and the probability of this conserved haplotype homozygously presenting in the Pakistani population would be very low. A functional impact on apoptosis consistent with a pathogenic mutation was demonstrated for purified neutrophils from Patients 1 and 4 using previously published assays (Smith et al, 2009). Both patients had a high level of spontaneous apoptosis (26·4%, 38·9% non-viable cells respectively at time zero compared to 3·6 ± 4·7% [mean±2SD] for 12 normal controls) (Fig 2). After 3 h incubation this level had significantly increased in Patient 1 compared to controls (58·2% versus 10·5 ± 7·1%) and was further enhanced in the presence of staurosporine (92·8% versus 12·9% ± 9·5% at 3 h). For Patient 4, the level of apoptosis only increased in the presence of staurosporine (36·4% without, 69·5% with staurosporine at 3 h). In addition, severe neutrophil glycosylation defects have been reported for Patients 1 and 3 (Hayee et al, 2011). Fig 2 Percentage of non-viable neutrophils in patients with the p.Thr64_Ile70del and p.Pro44Ser mutations, their carrier parents and controls. Graphical representation of the percentage of non-viable neutrophils (Annexin V-positive, propidium iodide-negative ... Overall, the frequency of G6PC3 mutations in our SCN cohort was 3% (4 of 155 patients/kindreds), although this incidence is likely to depend on the racial composition of the patient population investigated. No evidence has been found for a G6PC3 genotype-phenotype correlation (Banka et al, 2011b; Boztug et al, 2011), and it has been suggested that different founder mutations may exist according to racial groups (Banka et al, 2011b). Whether such variable genetic inheritance patterns has a wider impact on phenotype will require analysis of many cases, and the underlying reasons for the phenotypic heterogeneity associated with G6PC3 mutations are therefore currently unclear. Nevertheless, based on our observations of milder disease, isolated neutropenia and normal BM morphology but evident functional abnormalities in our two p.Pro44Ser-mutated cases, we would recommend G6PC3 screening as part of the work-up of all CN/SCN patients lacking other mutations, irrespective of the presence of additional congenital abnormalities.

EVALUATION OF RENAL FUNCTION IN TURKISH CHILDREN RECEIVING BFM-95 THERAPY FOR ACUTE LYMPHOBLASTIC LEUKEMIA

This study examined renal function in 42 children with acute lymphoblastic leukemia (ALL) treated according to BFM-95 protocol. Fifteen (group 1) were investigated longitudinally at 3 time points: before (T1), 4 weeks after (T2), and 2–6 months after (T3) consolidation therapy with high-dose methotrexate (HDMTX). The frequency of abnormalities in glomerular and tubular tests were nil at T1 and ranged from 13 to 40% at T2 and 7 to 33% at T3 in group 1. Twenty percent of the patients (n = 10) in group 2, who were examined at a single time point 7–36 months after consolidation, had glomerular and tubular abnormalities. There was only mild tubular abnormality in 5.8% of patients (n = 17) in group 3, who were examined at a single time point a mean of 56.1 ± 12.5 months after completion chemotherapy. These data show that consolidation therapy with HDMTX is frequently associated with acute renal toxicity in children with ALL but does not leave clinically significant late sequelae.

The Frequency of Menorrhagia and Bleeding Disorders in University Students

Study Objective: Menorrhagia is an important health problem in women of reproductive age. The aims of this study were to assess the prevalence of menorrhagia and hemostatic abnormalities associated with menorrhagia in university students. Methods: The pictorial blood assessment chart (PBAC) was used to identify students with menorrhagia. Those with a PBAC score > 100 were examined by pelvic ultrasound and laboratory tests including complete blood count, levels of clotting factors, von Willebrand factor antigen, and ristocetin cofactor activity and Platelet Function Analyser-100 (PFA-100). Platelet aggregation was studied in students with prolonged PFA-100 closure time. Results: Menorrhagia was identified in 82 (21.8%) of 376 students. Six of 82 students who had pelvic pathologies were excluded. Eleven (14.5%) of the remaining 76 students were found to have bleeding disorders, including von Willebrand disease in five (6.5%), platelet function disorder in four (5.2%), and clotting factor deficiencies in two (2.6%). Conclusions: Menorrhagia is a common but mostly unrecognized and untreated problem among university students. Underlying bleeding disorders are not rare and require comprehensive hemostatic evaluation for identification.

Candida vertebra osteomyelitis in a girl with factor X deficiency

Summary.  Candidal vertebra osteomyelitis is a rare condition which occurs primarily in immunocompromised patients. We report a 14‐year‐old girl with factor X deficiency who developed candida vertebra osteomyelitis during home therapy. The microorganism was probably from a contaminated peripheral cannula used for infusion of factor concentrate. This is the first such case in bleeding disorders to our knowledge.

A Novel Mutation in the SLC19A2 Gene in a Turkish Female with Thiamine-responsive Megaloblastic Anemia Syndrome

Reported here is a 2-year-old girl who was diagnosed to have thiamine-responsive megaloblastic anemia during evaluations for her bilateral neurosensorial deafness. Besides reporting a new mutation on the gene SLC19A2 for the first time in the literature, we highlight the recognition of this syndrome--when megaloblastic anemia and diabetes mellitus coexists--and the role of thiamine replacement for the treatment of both disorders.

Prognostic Factors and Long-Term Outcome in 52 Turkish Children With Hemophagocytic Lymphohistiocytosis.

Objectives: Hemophagocytic lymphohistiocytosis is a syndrome of pathologic immune activation that shares similar clinical and laboratory phenotypes with severe sepsis. Recent studies led to better recognition of hemophagocytic lymphohistiocytosis by clinicians, but no consensus exists on the criteria for high-risk patients. Design: We retrospectively reviewed the medical records of patients diagnosed with hemophagocytic lymphohistiocytosis to analyze the risk factors associated with poor outcome. Setting: Pediatric intensive care and hematology units of three tertiary hospitals in Turkey. Participants: Fifty-two children with hemophagocytic lymphohistiocytosis. Interventions: None. Measurement and Main Results: There were a total of 52 children meeting the diagnostic criteria of Histiocytic Society. Of them, 28 (54%) had a primary hemophagocytic lymphohistiocytosis. Mutation studies were performed in 18 of 28 patients (65%). Fourteen of them had PRF1, STX11, STXBP2, and UNC13D mutations, and four had Rab27a and LYST mutations. The remaining 24 patients (46%) were defined as having secondary hemophagocytic lymphohistiocytosis. Twenty-one of them had infection-associated hemophagocytic lymphohistiocytosis, and three had lysinuric protein intolerance. The mortality rate was significantly higher in primary hemophagocytic lymphohistiocytosis (64%) than in secondary hemophagocytic lymphohistiocytosis (16%) (p < 0.05). There were no significant differences for survival rate between hemophagocytic lymphohistiocytosis 94 (44%) and hemophagocytic lymphohistiocytosis 2004 (64%) protocols (p > 0.05). Age below 2 years, hyperferritinemia, thrombocytopenia, high disseminated intravascular coagulation score at diagnosis, and no clinical response at 2 weeks of treatment were independent prognostic factors for poor prognosis. Conclusions: Our data suggest that disseminated intravascular coagulation score greater than or equal to 5 can be used in the definition of high-risk patients. Early recognition of poor risk factors has important prognostic and therapeutic implications.