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.

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.

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.

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.

Epstein-Barr virus genotypes in AIDS-associated lymphomas are similar to those in endemic Burkitt's lymphomas.

PCR was used to screen EBV-positive lymphomas from endemic and sporadic Burkitt's lymphoma patients, including EBV-positive lymphomas derived from patients with HIV infection. Only 10% of sporadic lymphomas from either North America (1/15) or South America (2/14) were associated with the type 2 EBV strain, whereas 50% (8/16) of lymphomas from equatorial Africa and 46% (10/22) of HIV-associated lymphomas were positive for the type 2 strain. These data, in conjunction with previous reports, suggest that the proportions of strain types in Burkitt's lymphoma reflect the proportions of strain types in peripheral lymphocytes, and not simply the prevailing regional strain. The increased association of the type 2 strain in lymphocytes and lymphomas from HIV-infected individuals and from Africa may be a result of intermittent (malaria) or continuous (HIU) compromise of immune function in these populations.

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.

Is the Ala138Gly alteration of MEFV gene important for amyloidosis?

Progressive systemic amyloidosis is the most important complication of familial Mediterranean fever that inevitably leads to chronic renal failure. Initial studies have suggested that the presence of the Met694Val mutation carry a significant risk for the development of amyloidosis. On the contrary, our data revealed that there was no dominance of any MEFV mutation in relation to amyloidosis. The difference between our mutation data and others led us to study a polymorphism in Turkish population that might be a risk factor for the occurrence of amyloidosis. As some of the previously reported exonic polymorphisms in other disease states found to increase the genetic susceptibility, we aimed to study Ala138Gly of the MEFV gene. Our study group consisted of 124 FMF patients, of which 47 had amyloidosis. Eighty-one individuals without any familial history of FMF were included as control group. There was no statistically significant difference between healthy controls and FMF patients for the Ala138Gly polymorphism (p=0.9). However, when FMF/amyloidosis patients (n:47) were taken as another group, the difference was significant (p= 0.01) indicating that the carriers of 138Gly are more prone to amyloidosis [odds ratio 3.1 (CI 95% 1.57-5.75)].

MEFV mutations in Turkish patients suffering from Familial Mediterranean Fever.

Familial Mediterranean fever (FMF) is a recessive inherited disorder affecting Sephardic Jews, Arabs, Armenians and Turks. The gene responsible for FMF was recently cloned and several disease-associated mutations have been described. We have evaluated seven MEFV mutations in 460 chromosomes of 230 unrelated patients with FMF living in Turkey, using PCR methods. The M694V allele accounted for 43.5% of the alleles studied and 19.1% of the patients were homozygous. The M680I, V726A and M694I mutations were responsible for 12.0%, 11.1% and 2.8% of the patients respectively. R761H, K695R and E148Q were rarely encountered. Two thirds of the disease alleles were attributed to three common mutations: M694V, M680V and V726A, but only 54% of the patients carried one or two of the three mutations. Adding the four rarer mutations increased these figures to 72% and 60%, respectively. Altogether, 79.6% of the patients bore at least one of the main mutations, and 84.3% carried at least one of the seven mutations studied. The 28 patients suffering also from amyloidosis carried at least one of five mutations, M694V being the most common. These results suggest that the origin of FMF in Turkey is heterogenous, all common mutations are associated with amyloidosis. Further, rapid and accurate molecular diagnosis of FMF is feasible in most cases.

Isolated growth hormone (GH) deficiency type IA associated with a 45-kilobase gene deletion within the human GH gene cluster.

A Turkish family of seven individuals (two parents and five offspring) is described in which three children presented with isolated GH deficiency type IA, as defined by Illig et al. The gene deletion responsible for the isolated GH deficiency was characterized by Southern blotting and hybridization analysis of genomic DNA using a 32P-labeled hGH cDNA clone as a probe. In the affected patients, a total of approximately 45 kilobases of DNA, encompassing the human (h) GH-1, human chorionic somatomammotropin-L (hCS-L), hCS-A, and hGH-2 genes, were deleted. The end points of the deletion lay within two regions of highly homologous DNA sequence situated 5' to the hGH-1 gene and 5' to the hCS-B gene. The retention of only the hCS-B gene was associated with normal weight and length at birth and normal postpartum lactation in the mother heterozygous for the deletion. The parents, who are consanguineous, both presented with a DNA restriction pattern consistent with heterozygosity for this deletion.

Familial Mediterranean fever and systemic amyloidosis in untreated Turkish patients.

We compared the frequencies of seven MEFV mutations (M694V, M680I, V726A, M694I, K695R, R761H, E148Q) and the clinical findings in 20 Turkish FMF patients who had not developed amyloidosis by the age of 40 years in the absence of colchicine therapy, with those in 27 Turkish amyloidosis patients. No mutation frequency, including that of M694V, was different between the two groups. Family history of amyloidosis and parental consanguinity were noted to be higher in the amyloidosis group. The seven mutations do not appear to be sufficient to explain the development of amyloidosis in Turkish FMF patients. Other genetic factors may be important for this association.

Common mutations at the homocysteine metabolism pathway and pediatric stroke.

Heterozygosity and/or homozygosity for mutations at the genes of the enzymes involved in homocysteine metabolism may confer an increased risk for thrombosis by causing hyperhomocysteinemia. Although the mutations related to homocysteine metabolism possibly increase the risk of stroke, the data are conflicting and there are very few reports linking these defects to acute stroke in children. We aimed to study the role of these mutations in Turkish children with ischemic stroke. Forty-six patients having cerebral infarct were clinically diagnosed, and the infarction verified with magnetic resonance imaging of the brain was included in the study. All patients were below the age of 18 (10 months to 18 years). Sixty-eight controls, consecutively selected among healthy unrelated subjects from the same geographic area of Turkey without personal and family history of thrombosis, stroke or Behest's disease, were included. Genotyping for the common mutations was carried out by the methods described previously. There was no difference between the pediatric stroke patients and controls for the distribution of methylene tetrahydrofolate reductase (MTHFR) 677 C-T, MTHFR 1298 A-C, methylene tetrahydrofolate dehydrogenase (MTHFD) 1958 G-A and methionine synthase reductase (MTRR) 66 A-G alleles. There was no risk for double gene alterations (MTHFR 677 C-T vs. 1298 A-C) after individuals with FV 1691 A mutation is excluded. Twelve of the 46 patients were found to carry FV 1691 A mutation (26.0%), one being homozygote. The cerebral infarct risk for FV 1691 A was found to be 6.4 (CI 95% 1.7-23.0). Eight of the 46 patients were found to carry PT 20210 A mutation (16.6%). Two of the FV 1691 A heterozygous patients carried PT 20210 A mutation at the same time (4.2%). As a conclusion, we can say that FV 1691 A and PT 20210 A mutations are important and must be included to the routine analysis of pediatric stroke patients.

The alpha2 Gene alleles of the platelet collagen receptor integrin alpha2 beta1 in Turkish children with cerebral infarct.

The possible role of point mutations in the platelet integrin alpha2 beta1 gene in Turkish children with ischemic stroke was evaluated in this study. The case-control study included 44 pediatric patients with cerebral infarct (age range, 10 months to 18 years) and 96 healthy unrelated individuals. Genotyping was performed according to previously described methods. Distribution of the three haplotypes were 36.4%, 45.3%, 10.4% and 31.8%, 50.0%, 13.6% for the controls and the patients, respectively. A new fourth haplotype was found which was 7.8% and 4.5% respectively. Our data indicated that these haplotypes are not risk factors in pediatric stroke group.

The role of prothrombotic mutations in patients with Buerger's disease.

Thromboangiitis obliterans (TAO), or Buerger's disease, is a segmental occlusive inflammatory disorder of the arteries and veins, and etiopathogenesis is still obscure. In the present study we investigated the prevalence of prothrombin 20210 G-->A, factor V 1691 G-->A (Factor V Leiden), and factor V 4070 A-->G (His 1299 Arg) mutations, found to be associated with increased risk for vascular thrombosis, in 36 patients with TAO. We performed a case-control study of these mutations. The odds ratio for prothrombin 20210 A allele compared with G allele was 7.98 (95% confidence intervals 2. 45-25.93). Only this prothrombotic genetic factor was associated with the risk of TAO (p=0.032). In conclusion, carrying the prothrombin 20210 G-->A may be an important prothrombotic risk factor of TAO. This genetic predisposition must be screened in these patients routinely, and clinical importance must be supported by further investigations.

Effect of plasminogen activator inhibitor-1 4G/5G polymorphism in Turkish deep vein thrombotic patients with and without FV1691 G-A.

A decreased fibrinolytic activity due to increased levels of plasminogen activator inhibitor-1 has been shown in deep vein thrombosis patients. Elevated plasma plasminogen activator inhibitor-1 levels are associated with the 4G allele of a 4G/5G polymorphism located in the promoter region of the plasminogen activator inhibitor-1 gene. Because there is no existing data in the Turkish population, we aimed to study these mutations in patients with deep vein thrombosis (n = 136) and normal controls (n = 113), consecutively selected among unrelated healthy subjects without personal and familial history of atherothrombosis from Ankara, Turkey. DNA was extracted by conventional methods, and polymerase chain reaction of the plasminogen activator inhibitor-1 4G/5G polymorphism was performed according to a previously described method. Genotype distributions of FV 1691G-A and plasminogen activator inhibitor-1 4G/5G are as follows: plasminogen activator inhibitor-1 4G (patients) 0.562, plasminogen activator inhibitor-1 4G (controls) 0.50 (p = 0.6); FV1691A (patients) 0.147, FV1691A (controls) 0.035 (p = 0.005). Our data indicated that plasminogen activator inhibitor-1 4G/5G does not have an effect on the thrombotic risk. Carrying the 4G allele either in heterozygous or homozygous state increases the risk in the presence of FV1691A (odds ratio: 9.8 and 6.9, confidence interval 95% 2.9-32.7 and 1.3-35.8). FV1691A is an independent risk factor for thrombosis (odds ratio: 5.5, confidence interval: 95% 2.5-12.1). We concluded that coexistence of FV1691A and plasminogen activator inhibitor-1 4G allele leads to an increased risk for thrombosis leading a further evidence to another prothrombotic factor that may be necessary for the development of a manifest thrombotic event.

Effect of methylenetetrahydrofolate reductase 677 C-T, 1298 A-C, and 1317 T-C on factor V 1691 mutation in Turkish deep vein thrombosis patients.

Possible effect of three common mutations in (MTHFR 677 C-T; 1317 T-C; 1298 C-A) and FV 1691 G-A mutation was studied in Turkish patients with thrombosis and compared with normal controls. The case-control study included 68 patients with the diagnosis of deep vein thrombosis and 66 controls, consecutively selected among subjects without personal and familial history of atherothrombosis. Patients with deep vein thrombosis were selected if Doppler ultrasonography was positive. Only, the comparison of factor V 1691 G-A mutation revealed statistically significant difference in control (6.06%) and deep vein thrombosis (23.5%) group. Risk assessment of double prothrombotic gene alterations revealed only FV 1691 G-A mutation as an independent risk factor for thrombosis (odds ratio 4.7 [1.5-15.0]), but our data suggested that MTHFR 677 has effect on its own (odds ratio 1.97 [0.6-2.7]) but may have synergy with FV 1691 G-A (odds ratio 8.12 [2.0-25.3]). However, MTHFR 1298 A-C and 1317 T-C does not have any effect; furthermore, being heterozygote at two different loci or homozygosity at least in a locus for 677 and 1298 revealed a significant increase (odds ratio 9 and 24 [1.3-59.3 and 2.3-240.3]) between these two groups.

Search for genetic factors favoring thrombosis in Turkish population.

Common mutations in three genes (MTHFR 677 C-T; MS 2756 A-G; CBS Exon 8,844 ins 68) in homocysteine metabolism have been shown to cause increased plasma homocysteine levels thus causing a predisposition to thrombosis. FV 1691 G-A mutation, which is very common in the Turkish population, was also studied. As there is no existing data in the Turkish population, we aimed to study these mutations in patients with thrombosis and normal controls. The case-control study included 52 patients with the diagnosis of deep vein thrombosis (DVT) and 106 controls, consecutively selected among subjects without personal and family history of atherothrombosis. Patients with DVT were selected if Doppler ultrasonography was positive. The comparison of FV 1691 G-A mutation revealed statistically significant difference in control and DVT group. Risk assessment of double prothrombotic gene alterations indicated only FV 1691 G-A mutation as an independent risk factor for thrombosis, but our data suggested that MTHFR 677 has little effect on its own but may have synergy with FV 1691 G-A. Other possible risk genotypes at the homocysteine pathway did not have a significant effect on thrombosis. Furthermore, being heterozygote at two different loci or homozygosity at least in one locus also did not reveal a significant difference between these two groups in our population.

Familial Mediterranean fever (FMF)-associated amyloidosis in childhood. Clinical features, course and outcome.

OBJECTIVE: Familial Mediterranean fever (FMF) is an autosomal recessive disorder of childhood characterized by attacks of fever and serositis. Renal amyloidosis is the most important complication of the disease that determines the prognosis. METHODS: Forty-eight Turkish FMF patients with amyloidosis who have been followed at the two hospitals in Ankara were included in this study. RESULTS: All patients with amyloidosis had been symptomatic for FMF at the time of the diagnosis (Phenotype I), none had received regular colchicine therapy and all presented with proteinuria. Ten of them had asymptomatic proteinuria; 38 had nephrotic syndrome and 8 of them had renal insufficiency (CRI) as well, at the time of the diagnosis. Regular colchicine therapy was commenced to all of the patients. At the end of observation period of 4.5 +/- 2.23 years (range 2-12 yrs) on treatment, nephrotic syndrome resolved in 13 patients and proteinuria was lost in 5 of them. None but 2 of the patients who were diagnosed at proteinuric stage progressed to end stage renal failure (ESRF). Seven MEFV mutations (M694V, M680I, V726A, M694I, K695R, R761H, E148Q) were systematically investigated in 32 patients. Six of the seven studied mutations were found in these patients and clinical diagnosis was confirmed by mutation analysis in 24 patients. Eight patients were found to have mutations on one of the alleles. CONCLUSION: Amyloidosis is the most serious complication of FMF. Colchicine treatment ameliorates the progression of renal disease in the patients who presented with proteinuria and even with nephrotic syndrome. No correlation between the outcome of the patients with nephrotic syndrome and the degree of proteinuria and/or serum albumin levels at the initiation of treatment were noted. Progression to ESRF seems inevitable despite colchicine therapy after the development of CRI in patients with FMF associated amyloidosis.