Determination of CEBPA mutations by next generation sequencing in pediatric acute leukemia.

OBJECTIVES: The CCAAT/enhancer-binding protein-alpha (CEBPA) is lineage-specific transcription factor in the hematopoietic system. In this study, we aimed on the clinical features and the prognostic significance associated with CEBPA mutations in 30 pediatric patients with acute leukemia. METHODS: In addition, the association between found variants and mutations of Ten-Eleven-Translocation 2 (TET2), Kirsten rat sarcoma viral oncogene homolog (KRAS), and Casitas B-cell lymphoma (CBL), FLT3 (Fms-Related Tyrosine Kinase), JAK2 (Januse Kinase-2) and Nucleophosmin 1 (NPM1) were analyzed, which are important prognostic risk factors for pediatric acute leukemia patients. The entire CEBPA coding region was screened using the NGS method. RESULTS: CEBPA mutations were detected in 16 (53.3 %) of 30 patients. In total, ten distinct of nucleotide changes were identified in 30 patients, including 6 novel and 4 known mutations by sequencing the entire CEBPA gene. We found 6 frame shift mutations, 1 missense mutation, 3 synonymous variants. The most common mutation was the c.487del G resulting p.Glu163Ser in 5 cases. Three patients carried CEBPA double mutations. CONCLUSION: The detected variants in this article seemed to be the first screening results of genes studied by NGS in pediatric acute leukemia patients. Our results also showed some degree of association between FLT3-ITD, TET2, KRAS, CBL and CEBPA mutations (Tab. 4, Fig. 1, Ref. 24).

The role of prothrombotic factors in children with hemiplegic cerebral palsy.

AIM: Hemiplegic cerebral palsy (HCP) is a condition occurring as a consequence of a non-progressive damage of the brain with incomplete anatomical and physical development during the early period of life. Its etiology is multifactorial, with the cause remaining unexplained in the majority of cases. This study aims to investigate whether thrombophilic factors correlates with the etiology in children with HCP. METHODS: We included 36 children with HCP in the patient group, and 41 healthy children with no neurologic disorders in the control group. No significant difference was found between the two groups in terms of factor V leiden, methylenetetrahydrofolate reductase and prothrombin 20210A mutation frequency and protein C, protein S and antithrombin III levels. RESULTS: Homocysteine levels were significantly higher in the group of patients with HCP as compared to the control group (P=0.012). Because we could not identify the origin of hyperhomocysteinemia as congenital or acquired, the impact of hyperhomocysteinemia on HCP was considered insignificant. Each thrombophilic disorder was assessed in terms of relatedness to atrophy, periventricular leukomalacia, infarct, congenital anomaly and porencephalic cyst, respectively. No significant correlation was detected between thrombophilic disorders and cranial imaging findings. CONCLUSION: Our study has shown that thrombophilic factors are not involved in the etiology of HCP.

No significant association between the promoter region polymorphisms of factor VII gene and risk of venous thrombosis in cancer patients.

UNLABELLED: Factor VII (FVII) plays an important role in blood coagulation. The role of common polymorphisms influencing the FVII plasma levels in thromboembolic disorders has been evaluated but there is no published data related to the effect of FVII gene polymorphisms on the venous thrombosis risk in cancer. AIM: To investigate the association of three common functional polymorphisms in the promoter region of FVII gene: a decanucleotide insertion at position-323 (-323ins10-bp), a G to T substitution at position-401 (-401GT), and a G to A substitution at position-402 (-401GT) with venous thrombosis in cancer patients. MATERIALS AND METHODS: The study included 60 cancer patients with venous thromboembolism (VTE) (group 1) and 130 cancer patients without VTE (group 2). Genotyping of -323ins10-bp, -401GT, and -402GA polymorphisms in the promoter region of FVII gene was performed by the method of single-strand conformation polymorphism analysis and sequencing. Factor V Leiden (FVL) was also determined in all cases. RESULTS: The frequency of FVL was significantly greater in cancer patients with VTE compared with group 2 patients (p FVII gene, the distributions of genotypes and allele frequencies were not significantly different between two groups of patients ( p > 0.05). The results did not change significantly after the exclusion of patients carrying the FVL (p > 0.05). CONCLUSIONS: The screening for the -323ins10-bp, -401GT, and -402GA olymorphisms of FVII gene did not contribute to a meaningful diagnostic nvestigation in cancer patients with venous thrombosis.

Treatment challenges in pediatric stroke patients.

Aim. In this study we presented our experience of 18 years on the etiology, risk factors, prophylactic and acute treatment, the effect of treatment to recurrence rate of patients with stroke. Methods. The population included 108 patients who had been treated for stroke at Pediatric Neurology Department of Ankara University with the diagnosis of arterial ischemic stroke and sinovenous thrombosis between January 1992 and August 2010. Forty-one girls (38%) and 67 boys (62%) with mean symptom age 3.1 ± 4.04 years, (0-18 years old) were followed up with a mean period of 4.9 ± 3.78 years (0-17 years). Results. 30 patients had no risk factors, 34 patients had only one risk factor and 44 patients had multiple risk factors. Recurrence was seen in three patients. There was no any statistical correlation between the recurrence of stroke and the existence of risk factors (P = .961). Seventeen patients received prophylactic treatment; 2 of them without any risk factors, 3 had one risk factor, 12 patients, who constituted the majority of our patients, had multiple risk factors (P = .024). Conclusion. With this study we showed that the right prophylaxis for right patients reduces the rate of recurrence.

Disorders of sexual development: an overview of 18 years experience in the pediatric Endocrinology Department of Ankara University.

INTRODUCTION: Disorders of sexual development (DSD) occur when the appearance of the internal and/or external genitalia is at variance with normal development for either sex. We reviewed the characteristics of patients with DSD. PATIENTS: Two hundred and eight children aged from newborn to 19 years with DSD from 1990 to 2008. RESULTS: 46,XY DSD (52.4%) was more common than 46,XX DSD (34.6%) and gonadal differentiation disorders (12.99%). Thirty-six (33.02%) patients were diagnosed with androgen resistance syndrome, 41 (37.61%) had 5alpha-reductase deficiency, 23 (21.10%) had testosterone synthesis disorders. Congenital adrenal hyperplasia was the most frequent underlying cause of 46,XX DSD. CONCLUSION: There are many difficult aspects in the diagnosis and management of DSD. Gender assessment teams of endocrine centers need a multidisciplinary approach for the diagnosis, medical and surgical treatment, genetic counseling, and psychosocial support of these patients.

Factor V 1691 G-A (Leiden) polymorphism and cancer-related venous thromboembolism: a meta-analysis of published studies.

PURPOSE: Although one of the most common genetic polymorphisms that predispose to venous thromboembolism (VTE) is factor V 1691 G-A (Factor V Leiden; FVL), the effect of this polymorphism on the development of VTE in cancer patients is unclear. We have therefore performed a meta-analysis to estimate the risk of VTE associated with FVL among cancer patients. MATERIALS AND METHODS: Relevant studies published before May 2006 were retrieved from Pubmed/Medline. We selected studies comparing the prevalence of FVL in cancer patients with VTE with cancer patients without VTE. Both fixed and random effect models were used. P-values and odds ratios (ORs) with 95% confidence intervals (95% CI) were calculated by Fisher's exact test. RESULTS: Pooled results from 9 studies, comprising 397 cancer patients with VTE and 678 cancer patients without VTE revealed that the prevalence of FVL was higher among cancer patients with VTE (7.3%) than those without VTE (4.6%) (chi(2) = 34.633 > chi(2) (18; 0.05) =28.869) (p=0.013). It was also found that mean effect size of FVL was 0.22 (95% CI 0.051-0.4892). Using the homogeneity test, there was evidence of statistical heterogeneity (Q(hom)= 46.334> chi(2) (8; 0.05) =15.507) (p=0.0000). CONCLUSION: Although the published studies were small and the meta-analysis demonstrated an association of FVL with cancer-related thrombosis, testing for FVL can be routinely used as part of clinical thrombophilia assessment in cancer patients from the regions with high incidence of FVL.

Chromosome 22q11.2 deletion presenting with immune-mediated cytopenias, macrothrombocytopenia and platelet dysfunction.

OBJECTIVE: To report a case of chromosome 22q11.2 deletion presenting with large platelets, platelet dysfunction, immune-mediated thrombocytopenia and neutropenia, in addition to other features of the disease. CASE PRESENTATION AND INTERVENTION: The patient presented in the neonatal period with tetralogy of Fallot, subtle dysmorphic features and thrombocytopenia. Fluorescent in situ hybridization analysis confirmed the diagnosis of chromosome 22q11.2 deletion. Further investigations showed immune thrombocytopenia and neutropenia in addition to reduced expression of platelet GPIb and abnormal platelet aggregation studies. CD4:CD8 ratio was reversed. His cardiac abnormality was successfully corrected surgically. He had mild recurrent bacterial infections. Recurrent epistaxis was becoming increasingly more severe, and he had cognitive developmental and speech delay. His serum calcium, phosphorus and parathormone have remained normal. CONCLUSIONS: Immune thrombocytopenia can coexist with macrothrombocytopenia and platelet dysfunction in chromosome 22q11.2 deletion and may present with significant bleeding episodes.

Sequencing of the factor 8(F8) coding regions in 10 Turkish hemophilia A patients reveals three novel pathological mutations, and one rediagnosis of von Willebrand's disease type 2N.

The most common cause for severe cases of hemophilia A is the homologous recombination involving intron 22 and related sequences outside the F8 gene. F8 coding regions of the gene including the exon/intron junctions were sequenced in 10 Turkish hemophilia A patients all of whom have been typed negative for intron 22 inversion and who did not have a detectable change by DGGE analysis. Pathological changes including two novel deletions (c. 205del CT and c. 3699del ACAT), one novel missense mutation (9546A) and two recurrent missense mutations were observed in five patients. The c. 2110C > T is another novel pathological change affecting exonic splicing enhancer site in two patients. One of the remaining three patients had a recurrent vWD type 2N mutation in the F8 binding site of the vWF (C788R). The S1269S polymorphism (c. 3864A > C) detected phenotype. Conclusively, sequencing of the promoter and the coding regions of 10 hemophilia A patients contributes four novel pathological mutations to the F8 mutations list and reveals a rediagnosis of hemophilia A but is still not sufficient to confirm hemophilia A phenotype in two patients.

Plasminogen activator inhibitor-1 (PAI-1) gene polymorphism (-675 4G/5G) associated with obesity and vascular risk in children.

UNLABELLED: Atherothrombotic complications in insulin resistance are partly attributed to impaired fibrinolysis caused by increased PAI-1 plasma levels, and 4G/5G promotor polymorphism of the PAI-1 gene may modulate PAI-1 transcription. OBJECTIVE: To investigate PAI-1-675 4G/5G allele gene polymorphism and its relationship with obesity in children. CHILDREN AND METHOD: The study participants were 133 apparently healthy non-obese children, 24 probable exogenously obese without family history (Group I), 66 probable familial obese (Group II), and 44 obese children who were referred to the pediatric endocrinology department with any complication of obesity (Group III). Group I and Group II obese children were gathered from a school-based epidemiological study. RESULTS: Incidence of obesity was 19% in a school with high socio-economic status, whereas it was 4% in a school with low socio-economic status. Frequencies of 4G/4G gene polymorphisms were 24.81%, 37.50%, 64.80% and 61.11% in the control group, and groups I, II, and III, respectively. In groups II and III, 4G/4G gene polymorphism, and in non-obese control children 5G/5G gene polymorphism, was common. In obese children in the presence of family history for obesity and metabolic syndrome (odds ratio [OR]: 4.48, 95% confidence interval [CI]: 1.26-15.82), carriage of the 4G allele either in heterozygous or homozygous state increased the risk of vascular disease (OR: 6.10, 95% CI 1.64-22.90). In patients with acanthosis nigricans, high HOMA-IR values, hypertriglyceridemia and elevated atherogenic index, 4G/4G genotype frequency was remarkably higher compared to patients with other features of metabolic syndrome. CONCLUSION: The increasing prevalence of childhood obesity in high socio-economic status is associated with health risks. In obese children with family history of obesity and cardiovascular disease or type 2 diabetes mellitus and in obese children who had any feature of metabolic syndrome, frequency of 4G/4G genotype was more than the 4G/5G and 5G/5G genotypes in the PAI-1 gene. These patients can be at increased risk for developing vascular disease. Acanthosis nigricans, high HOMA-IR value, hypertriglyceridemia and high atherogenic index can also reflect the high risk of vascular disease in metabolic syndrome.

Detection of virulence factors of Escherichia coli from children by multiplex polymerase chain reaction.

A total of 161 Escherichia coli (E. coli) strains isolated from children with urinary tract infection (UTI) were analysed for the genes encoding the virulence factors such as pyelonephritis (pap), s fimbriae (sfa), afimbrial adhesin I (afaI), haemolysin (hly), cytotoxic necrotising factor I (cnf I) and aerobactin (aer) by multiplex PCR. Ninety-four E. coli strains were found to carry at least one virulence factor. Therefore, 58.38% of total population was positive for one virulence gene at least. Percentage of genes within the total population for pap, sfa, afaI, hly, cnf I and aer was found as 22.98, 6.21, 9.94, 1.24, 9.94 and 39.75, respectively. Our analysis showed that sfa-pap (p < 0.001); pap-aer, afaI-aer and cnf I-pap (P < 0.05) and hly-sfa (p < 0.01) significantly co-occurred in their respective samples. In the light of these findings, we suggest an important role of pap causing UTI.

Plasminogen activator inhibitor -1 gene 4G/5G polymorphism in patients with breast cancer.

PURPOSE: The plasminogen activator inhibitor-1 (PAI-1) plays an important role in the development of tumor invasion and metastasis. The 4G allele of 4G/5G insertion / deletion polymorphism located in the promoter region 675 bp upstream from the transcription start sequence of PAI-1 gene is responsible for the higher plasma PAI-1 level. The aim of the present study was to evaluate the association between PAI-1 gene 4G / 5G polymorphism and breast cancer. MATERIALS AND METHODS: Two groups were investigated: the first group(group 1)was composed of 34 patients with breast cancer, the second group (group 2) consisted of 90 unrelated healthy women without history of malignancy. Genomic DNA isolation was performed from peripheral venous blood by standard phenol-chloroform extraction and polymerase chain reaction of the PAI-1 4G/5G polymorphism was performed. RESULTS: The prevalence of 4G/4G or 4G/5G genotype in group 1 and 2 was 97.1% and 78.8%, respectively. The distribution of the PAI-1 4G/5G genotypes was significantly different between the two groups (p<0.05). CONCLUSIONS: We suggest that this polymorphism may contribute to an inherited predisposition to the development of breast cancer, however further studies with larger series from diverse ethnic populations are needed to confirm our results.

Subtypes of genotype A Candida albicans isolates determined by restriction endonuclease and sequence analyses.

Systemic yeast infections are the Leading cause of mortality and morbidity in immunocompromized patients. Candida albicans, being the most frequently isolated fungal pathogen in these patients, can be divided into three genotypes (genotypes A, B and C) by 25S intron analysis. In our study, we found that molecular sizes of genotype A C. albicans isolates were heterogeneous. In order to determine the molecular basis of this difference, Haelll digestion was applied, and strains forming different band patterns were analyzed by automated sequence analysis. As a result of sequence analysis, eight different subtypes (a --> h) were found among genotype A C. albicans strains and an easy differentiation scheme consisting of Haelll and MspI digestions was constructed.

Evidence for single origins of 35delG and delE120 mutations in the GJB2 gene in Anatolia.

Eighteen different sequence changes, including three novel alterations, were detected in GJB2, encoding connexin 26, in 371 Turkish probands with non-syndromic sensorineural hearing loss. Two frequently detected mutations, 35delG and delE120, were shown to have single origins based on the conserved genotypes of two closely linked microsatellite and five single nucleotide polymorphism markers. Carrier frequencies of 35delG and delE120 in Egypt and Turkic populations of the Near East provide insights about the origin of these two mutations.

Genotype distribution of Candida albicans isolates by 25S intron analysis with regard to invasiveness.

The aim of this study was to genotype Candida albicans strains isolated from patients with invasive and non-invasive deep-seated infections. For this purpose, 301 C. albicans isolates (81 invasive and 220 non-invasive) were genotyped by using specific PCR primers designed to span the transposable group I intron of the 25S rDNA gene. Fifty-three of the 81 invasive isolates were genotype A (65.4%), eight were genotype B (9.9%) and 20 were genotype C (24.7%), while 98 of the 220 non-invasive isolates were genotype A (44.6%), 46 were genotype B (20.9%) and 76 were genotype C (34.5%). Genotype A was more prevalent among invasive isolates and genotypes B and C were more prevalent among non-invasive isolates (P = 0.0046). Genotypes D and E which represent C. dubliniensis were not found. These results indicate that there may be a relationship between C. albicans genotypes and invasiveness; genotype A being more invasive than others. The presence or absence of the transposable group I intron in the 25S rDNA gene may be important in determining the invasiveness of C. albicans.