Both Granulocytic and Non-Granulocytic Blood Cells Are Affected in Patients with Severe Congenital Neutropenia and Their Non-Neutropenic Family Members: An Evaluation of Morphology, Function, and Cell Death

Objective: To examine granulocytic and non-granulocytic cells in children with severe congenital neutropenia (SCN) and their non-neutropenic parents. Materials and Methods: Fifteen patients with SCN and 21 non-neutropenic parents were evaluated for a) CD95, CD95 ligand, annexin V, propidium iodide, cell cycle, and lymphocyte subsets by flow cytometry; b) rapid cell senescence (of leukocytes) by senescence-associated ß-galactosidase stain; c) aggregation tests by aggregometer; d) in vitro bleeding time by PFA-100 instrument; e) mepacrine-labeled dense granule number of thrombocytes by fluorescence microscope; and f) hematomorphology by light and electron microscope. HAX1, ELANE, G6PC3, CSF3R, and JAGN1 mutations associated with SCN were studied in patients and several parents. Results: Significant increase in apoptosis and secondary necrosis in monocytes, lymphocytes, and granulocytes of the patients and parents was detected, irrespective of the mutation type. CD95 and CD95 ligand results implied that apoptosis was non-CD95-mediated. Leukocytes of 25%, 12.5%, and 0% of patients, parents, and controls showed rapid cell senescence. The cell cycle analysis testable in four cases showed G1 arrest and apoptosis in lymphocytes of three. The patients had HAX1 (n=6), ELANE (n=2), G6PC3 (n=2), and unidentified (n=5) mutations. The CD3, CD4, and NK lymphocytes were below normal levels in 16.6%, 8.3%, and 36.4% of the patients and in 0%, 0%, and 15.4% of the parents (controls: 0%, 0%, 5.6%). The thrombocytes aggregated at low rates, dense granule number/thrombocyte ratio was low, and in vitro bleeding time was prolonged in 37.5%-66.6% of patients and 33.3%-63.2% of parents (vs. 0% in controls). Under electron and/or light microscope, the neutrophils, monocytes, lymphocytes, and thrombocytes in the peripheral blood of both patients and parents were dysplastic and the bone marrow of patients revealed increased phagocytic activity, dysmegakaryopoiesis, and necrotic and apoptotic cells. Ultrastructurally, thrombocyte adhesion, aggregation, and release were inadequate. Conclusion: In cases of SCN, patients' pluripotent hematopoietic stem cells and their non-neutropenic parents are both affected irrespective of the genetic defect.

The first report of a homozygous codons 9/10 (+T) ß-thalassemia mutation in a Turkish patient.

For the first time in Turkey, we report a thalassemic patient with a homozygous codons 9/10 (+T) genotype. Currently, the patient is 3 years and 2 months old and received an initial transfusion at the age of 18 months. After being alloimmunized following this transfusion, he required frequent transfusions, every week to every other week. Although alloimmunization was controlled after methyl-prednisolone, intravenous immunoglobulin, plasmapheresis and rituximab, the transfusion requirements continued related to hypersplenism. Subsequent to splenectomy, transfusion requirements disappeared with average hemoglobin (Hb) levels around 11.0 g/dL. The mother underwent prenatal diagnosis (PND) when she became pregnant for the third time; this revealed a heterozygous codons 9/10 fetus.

Cytotoxic effects of etephon and maleic hydrazide in Vero, Hep2, HepG2 cells.

The toxicity of etephon and maleic hydrazide, used as plant growth regulators in agriculture, were reported as low in mammals in previous studies. However, in vitro cytotoxicity studies in mammalian cells are currently missing to understand their toxicity at molecular level. In the current study, the cytotoxicity of these compounds, were studied in Vero (African green monkey kidney epithelium), HepG2 (human hepatocellular carcinoma), Hep2 (human epidermoid cancer) cells by MTT ((3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolium bromure) and LDH (lactate dehydrogenase) assays. Maleic hydrazide had lower IC50 values for all cell lines compared to ethephon. Least cytotoxic effect treated by ethephon were observed in Vero, followed by HepG2 and Hep2. Similarly maleic hydrazide also showed least cytotoxicity on Vero cells, followed by Hep2 and HepG2 cells (p < 0.05). IC50 values in general were found to be highest in Vero cells, followed by HepG2 and Hep2 cells (p < 0.05). LDH and MTT assays showed correllation and had close relation except HepG2-maleic hydrazide application with the correlation coefficient for all >0.868 (p < 0.05). This study is expected to be a basis to understand the cytotoxic effects of ethephon and maleic hydrazide in mammal cells to be supplemented by further studies.

Recurrent macrophage activation syndrome associated with heterozygous perforin W374X gene mutation in a child with systemic juvenile idiopathic arthritis.

BACKGROUND: Recurrent macrophage activation syndrome (MAS) is rarely reported. AIM: To describe recurrent MAS in a 2.5-year-old girl with systemic juvenile idiopathic arthritis and heterozygous perforin mutation, which may have a role in the patient's first recurrence despite use of the HLH-2004 treatment protocol. OBSERVATIONS: In the presented case, MAS was initially controlled after the addition of etoposide to the treatment regimen. However, recurrence occurred 6.5 months after cessation of the HLH-2004 protocol. Subsequent recurrences may have occurred because of the family's noncompliance with treatment. CONCLUSIONS: The patient's extremely high serum ferritin level (267,054 ng/mL) and the recurrent course of MAS may have been because of the coexistence of juvenile idiopathic arthritis and heterozygous perforin W374X mutation. We suggest to search for mutations in HLH genes in recurrent MAS cases.

Smaller hippocampus volume is associated with short variant of 5-HTTLPR polymorphism in medication-free major depressive disorder patients.

AIM: Serotonin is known for its importance in the pathophysiology of major depressive disorder. Although the hippocampus is one of the key regions in which neurogenesis occurs, and serotonin plays an important role in neurogenesis, results of studies that investigate effect of the 5-HTTLPR polymorphism on hippocampal volumes in major depressive disorder are inconclusive. METHOD: We looked for a relationship between the 5-HTTLPR polymorphism and hippocampal volumes in 44 depressed patients (mean age ± SD 33.6 ± 9.5 years) and 43 healthy controls (30.4 ± 6.7 years). Region of interest analysis was conducted on the images acquired via MRI. RESULTS: Although hippocampal volumes were similar in healthy and patient groups, there was a significant interaction between genotype and diagnosis on hippocampus volumes. Post-hoc ANCOVA showed that hippocampal volumes of S/S homozygous depressed patients were smaller compared to healthy controls in both hemispheres. CONCLUSION: The 5-HTTLPR polymorphism has an effect on hippocampal volumes of depressed patients, which is apparent only in S/S genotype. It seems that decreased neurogenesis by effects of reduced serotoninergic transmission may be responsible for smaller hippocampal volumes observed in S/S homozygous depressed patients.

Plasminogen activator inhibitor I 4G/5G polymorphism in neonatal respiratory distress syndrome.

Fibrin monomers inhibit surfactant function. 4G/5G insertion/deletion polymorphism plays an important role in the regulation of plasminogen activator inhibitor 1 (PAI-1) gene expression. To examine the genotype distribution of PAI-1 polymorphism in 60 infants with respiratory distress syndrome (RDS) and 53 controls, an allele-specific polymerase chain reaction (PCR) was used. The proportion of 4G/4G, 4G/5G, and 5G/5G genotypes did not differ statistically between the RDS and control groups (P > .05). Having PAI-1 4G/4G genotype polymorphism appears to increase the risk of RDS (odds ratio [OR] =1.5; 95% confidence interval [CI], 0.5-4.3), although it was not statistically significant. No relation was found between the PAI-1 4G/5G polymorphisms and RDS, but there was an increased risk associated with the 4G variant of the PAI-1 gene. We believe that our findings of increased 4G allele of the PAI-1 gene in infants with RDS would also help to clarify the pathogenesis of RDS.

The frequency of A91V in the perforin gene and the effect of tumor necrosis factor-α promoter polymorphism on acquired hemophagocytic lymphohistiocytosis.

OBJECTIVE: Numerous acquired etiological factors, such as infections, malignancies, and collagen tissue disorders, are involved in the development of acquired hemophagocytic lymphohistiocytosis (AHLH). Not everyone with the same etiological factors developments AHLH, which suggests the role of additional genetic or environmental predisposing factors that remain to be identified. METHODS: Perforin gene A91V missense transition (C>T change at position 272 in exon 2 of the perforin gene) and TNF-α gene promoter-1031 T>C nucleotide substitution are 2 candidate genetic predisposing factors due to their potential to alter inflammatory responses. In the present study these changes were investigated in healthy controls and AHLH patients. RESULTS: A91V transition was observed in 7 of the 159 (4.4%) controls. Among the 44 AHLH patients, 5 (11.3%) were heterozygous and the difference in the frequency of A91V transition, although striking (odds ratio: 2.8), was not statistically significant (p=0.09). All A91V-positive patients had infection. TNF-α-1031 T>C polymorphism was examined in 164 healthy controls and 40 AHLH patients, and the CC risk-elevating genotype was noted in 7 (4.3%) of the controls and 1 (2.5%) of the AHLH patients. The frequency of C and T alleles was 22.5% (n=18) and 77.5% (n=62) among the AHLH patients, and 22% (n=72) and 78% (n=259) among the controls, respectively. There wasn't a statistically significant difference between the groups in terms of allele frequencies (p>0.05). CONCLUSION: The present results indicate that compared to controls, A91V mutation was 2.8-fold more prevalent (according to the odds ratio) in the AHLH patients. A91V mutation is not uncommon in the general population and increases the risk of AHLH in patients with an underlying condition, especially those with an underlying infection.

Assessment of clinical and laboratory presentations of familial hemophagocytic lymphohistiocytosis patients with homozygous W374X mutation.

Homozygous W374X mutation was identified in unrelated 13 patients (6M/7F) from consanguineous families, 62% of which had history of deceased sibling. Haplotype analysis provided evidence for the probable existence of a founder effect. Age at disease onset ranged from 1 day to 5.5 months (median 2 months). Hepatic dysfunction was observed in 69%, ascite 62%, hypertriglyceridemia 77%, each hyperferritinemia and hypofibrinogenemia 85%, CNS involvement 46% of patients while birth weights were in normal range. Those with very high ferritin (>20,000ng/ml) had extremely low fibrinogen levels. Two-thirds of patients receiving HLH protocol died within 20 days of therapy.

The effect of depression, BDNF gene val66met polymorphism and gender on serum BDNF levels.

OBJECTIVE: To determine the effect of BDNF gene val66met polymorphism on serum BDNF levels in drug-free patients with major depressive disorder (MDD) and healthy subjects, that differ by gender. METHODS: Sixty-six drug-free patients (19 males+47 females) with non-psychotic MDD and fifty-six healthy controls (18 males+38 females) were recruited. Three-way ANOVA was employed to analyze the effect of mental health status, met-carriage and gender on Hamilton Depression Rating Scale (HDRS) scores and serum BDNF levels, by using the MIXED Procedure (SAS). RESULTS: Patients had a lower serum BDNF level than healthy subjects (22.47 vs. 27.49; p<0.0001). Met-carrier patients had a higher HDRS score than Val homozygote's (25.99 vs. 22.99, p<0.02). Serum BDNF level for met-carrier subjects (patients+controls) was lower than Val homozygote subjects (23.08 vs. 26.87; p<0.002). However, there were no effects of two-way interactions of met-carriage and mental health status on HDRS scores and serum BDNF levels. There was no gender effect on HDRS scores in the patients. Overall, male subjects (patients+controls) had a higher serum BDNF level than female subjects (26.87 vs. 23.08; p<0.002). However, there were no effects of two-way interactions of gender with mental health status and met-carriage on serum BDNF levels. CONCLUSIONS: We replicated the previous findings of lower serum BDNF levels during depression and in females. In addition, we found that met-carriage had an effect in reducing serum BDNF levels, regardless of gender and depression. Further animal and human studies with a larger sample size should investigate whether BDNF val66met polymorphism could alter brain and serum BDNF levels.

Excessive naked megakaryocyte nuclei in myelodysplastic syndrome mimicking idiopathic thrombocytopenic purpura: a complicated pre- and post-transplantation course.

A boy 3 years 7 months old with thrombocytopenia and history of intracranial hemorrhage who underwent bone marrow transplantation is presented. He was refractory to steroids, immunoglobulin G, vincristine, azathioprine, cyclosporine A, interleukin-11, chemotherapy, and splenectomy. Idiopathic thrombocytopenic purpura was excluded by light /electron microscopic and flow cytometric findings; the diagnosis of refractory cytopenia, a subgroup of pediatric myelodysplastic syndrome, was made. Naked megakaryocyte nuclei were 55.38 +/- 28.2% vs. 31.67 +/- 23.22% of all megakaryocytes in the patient and the control group of 9 patients with idiopathic thrombocytopenic purpura, respectively (p = .016). The posttransplatation course was complicated by delayed platelet engraftment, bronchiolitis obliterans associated with pneumocystis carinii pneumonia, which resolved completely.

ALX4 dysfunction disrupts craniofacial and epidermal development.

Genetic control of craniofacial morphogenesis requires a complex interaction of numerous genes encoding factors essential for patterning and differentiation. We present two Turkish families with a new autosomal recessive frontofacial dysostosis syndrome characterized by total alopecia, a large skull defect, coronal craniosynostosis, hypertelorism, severely depressed nasal bridge and ridge, bifid nasal tip, hypogonadism, callosal body agenesis and mental retardation. Using homozygosity mapping, we mapped the entity to chromosome 11p11.2-q12.3 and subsequently identified a homozygous c.793C-->T nonsense mutation in the human ortholog of the mouse aristaless-like homeobox 4 (ALX4) gene. This mutation is predicted to result in a premature stop codon (p.R265X) of ALX4 truncating 146 amino acids of the protein including a part of the highly conserved homeodomain and the C-terminal paired tail domain. Although the RNA is stable and not degraded by nonsense-mediated RNA decay, the mutant protein is likely to be non-functional. In a skin biopsy of an affected individual, we observed a hypomorphic interfollicular epidermis with reduced suprabasal layers associated with impaired interfollicular epidermal differentiation. Hair follicle-like structures were present but showed altered differentiation. Our data indicate that ALX4 plays a critical role both in craniofacial development as in skin and hair follicle development in human.

Transient depletion of innate immunity in varicella infections in otherwise healthy children.

OBJECTIVE: Varicella is a common childhood infection and has a number of complications in the unvaccinated population. Perforin, found in natural killer cells, is important for the killing of virally infected cells. For this reason, the aim of this study was to determine natural killer cell count and activity, perforin expression, and Fas and soluble Fas ligand (sFas-L) levels in immunocompetent children with varicella infection and define any possible relations between the levels and varicella complications. METHODS: Forty children were analyzed at diagnosis and on the 15th day of varicella infection. There was a significant difference in hemoglobin levels and leukocyte and platelet counts between days 0 and 15. RESULTS: Thirteen (32%) patients were found to be lymphopenic. Natural killer cell count and activity were significantly higher on day 15 when compared to values at diagnosis. The Fas-mediated apoptotic pathway was found to be active in acute varicella infection because Fas and sFas-L levels at diagnosis were higher than values on day 15. CONCLUSION: These findings suggest that the Fas and Fas-L apoptotic pathway is active during the acute phase of the viral infection and that it becomes inactive by day 15, paralleling the hematologic recovery.

Placental fas/fas ligand expression in early pregnancy losses.

PROBLEM: The aim of this study was to compare the expression levels of Fas and Fas ligand (FasL) in first-trimester placentas obtained from spontaneous abortions in patients with antiphospholipid antibody syndrome (APS) or factor V (FV) Leiden mutation, compared with values in placentas from induced abortions in patients negative for these conditions. METHOD OF STUDY: We studied explants from 6- to 10-week-old placentas that had been prepared by collagenase digestion from 10 spontaneous abortions from APS-positive patients, nine spontaneous abortions in patients positive for FV Leiden mutation, and 10 induced abortions. All tissues were analyzed by flow cytometry for expression of Fas and FasL. RESULTS: Flow cytometric analysis showed that placental FasL expression was significantly lower in abnormal pregnancies than in normal ones. However, no such difference was observed for Fas expression. CONCLUSION: FasL on placental cells may be involved in the maintenance of immune privilege, thereby ensuring the safety and growth of placental tissues. Dysregulation of apoptotic mechanisms may play a critical role in spontaneous abortions.

Clinical and molecular aspects of Turkish familial hemophagocytic lymphohistiocytosis patients with perforin mutations.

The aim of this study was to elucidate the pathologic sequence changes and associated clinical phenotypes in 9 new patients showing homozygosity for perforin gene among a total of 37 (24%) Turkish FHL families studied by linkage analysis. These 9 unrelated patients (5M/4F) were coming from consanguineous families and their presentation ages of systemic symptoms were ranged from birth to 15 years. Direct sequencing of coding exons of the perforin gene led to the identification of five different homozygous alterations. The nonsense W374X mutation was identified in three patients while four different missense mutations namely G149S, V50M, A91V and novel A523D were detected in the rest six patients.

Neonatal primary hemophagocytic lymphohistiocytosis in Turkish children.

Although the data on hemophagocytic lymphohistiocytosis (HLH) has gradually increased, the neonatal-onset HLH patients have usually been reported as case reports or together with other age groups of patients. The aim of this study was to draw attention to the clinical and laboratory characteristics of neonatal HLH cases. Herein, the data of 8 primary, neonatal-onset HLH patients are reported. Mutational analyses were performed in 7 of the patients and mutations in UNC13D gene were detected in 3 of the patients, whereas 2 patients were found to have perforin gene mutation. Four of the patients were symptomatic within the initial 10 days of life. One patient with perforin mutation (1122 G>A) had a very severe clinical course and died on the seventh day of life before receiving any specific treatment. Another patient with UNC13D 2783 G>C, who became symptomatic on the sixth day of life, underwent early hematopoietic stem cell transplantation and is currently alive at 8 years of age. Two of these 4 patients had extensively high serum ferritin levels mimicking neonatal hemochromatosis. Of the 4 patients who became symptomatic after 20th day of newborn period, 1 was found to have perforin gene mutation (445 G>A) and 2 siblings were detected to have a missense mutation in UNC13D (640 C>T) gene. The latter patients with UNC13D mutations could survive 3 and 4 months, although their parents ceased therapy. The patient with perforin mutation survived 11 months.

Rapid cell senescence and apoptosis in lymphocytes and granulocytes and absence of GM-CSF receptor in congenital dysgranulopoietic neutropenia.

A girl with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother with aphthae (A) were investigated. Apoptosis in lymphocytes and granulocytes of both patients (mother A+) were documented by high annexin and electron microscopic morphology. CD11b/CD18 of the daughter's granulocytes ranged between low to normal while that of the mother changed between very low to high levels through A(-) to A(+) periods. In both patients, CD11b/CD18 on lymphocytes were high; GM-CSF receptor was negative; CD4-/CD8- lymphocytes were high and the leukocytes which showed abnormal cell cycle were stained by senescence associated beta-galactosidase. We think that increased apoptosis and rapid cell senescence of leukocytes underlies the pathophysiology of CDN.

Omeprazole inhibits natural killer cell functions.

This study was designed to determine the possible effects of omeprazole on human natural killer cells. Peripheral venous blood samples were taken from 20 peptic ulcer patients before and at the 14th and the 28th days of omeprazole treatment. Mononuclear cells were removed from blood and their capability of making conjugation with K562 target cells and lysing K562 target cells was evaluated. A significant decrease was found (P < 0.001) in the 14th and the 28th days compared with the basal value of the capability of the mononuclear cells to conjugate with the K562 target cells and to lyse them. This study demonstrated that omeprazole significantly reduces natural killer cell functions. This finding suggests that omeprazole may also have some effects on the other systems in addition to parietal cell acid secretion.

Congenital dysgranulopoietic neutropenia.

We investigated a 15-year-old female with congenital dysgranulopoietic neutropenia (CDN) and her non-neutropenic mother who had recurrent stomatitis. In both patients, cells of the neutrophilic, eosinophilic, monocytic, megakaryocytic, and basophilic series were dysmorphic. Plasmacytoid lymphocytes and mild megaloblastic erythroid precursors were present. Bleeding times of both patients were prolonged. The mother had a secondary aggregation defect; the number of the plasmacytoid lymphocytes, dense granules of platelets, and dysmorphic neutrophils, neutrophil chemotaxis, and myeloperoxidase content fluctuated according to the presence or not of aphthae. The daughter's karyotype revealed 46,XX/46,XX, t(1;8). No ELA2 or G-CSFR mutation was detected. These findings support stem cell involvement in CDN.

Effect of short course high dose methylprednisolone on endothelin-1 and nitric oxide in children with acute lymphoblastic leukemia: a preliminary study.

We have investigated serum endothelin-1 (ET) and nitric oxide (NO) levels before and after a short course of high dose methylprednisolone (HDMP) in children with acute lymphoblastic leukemia (ALL) as an indicator of vasoconstrictor and vasodilator properties of endothelium. Nineteen children with ALL (aged 13-180 months; 5 girls and 14 boys) and 25 healthy children were included in the present study. The children with ALL were given HDMP (20 mg kg 1 day 1) alone for the first 5 days. Serum ET and NO levels were analysed before and after a short course of high dose methylprednisolone. Before treatment, serum ET levels (median 7.6 pg ml 1) of the patients were lower than the healthy controls (13.0 pg ml 1) (p < 0.05), and it rose to similar levels (13.0 pg ml 1) following therapy as in the controls. Nitric oxide levels (7.0 micromol) of the patients were insignificantly higher than the healthy controls (3.9 micromol) and did not differ after treatment (7.0 micromol) (p > 0.05). In conclusion, the elevation of ET to normal level following treatment suggests that a short course of high dose of methylprednisolone improve the endothelial dysfunction caused by acute leukemia.