ß-Globin Gene Mutations in Pediatric Patients with ß-Thalassemia in the Region of Çukurova, Turkey.

ß-Thalassemia (ß-thal) is one of the most common genetic disorders in Turkey. In this study, we investigated the mutations and frequency of ß-thal at the molecular level in pediatric ß-thal patients in the Çukurova region. The ß-thal mutations of 52 cases were analyzed. An automated blood cell counter was used for hematological data. Cellulose acetate electrophoresis and high performance liquid chromatography (HPLC) methods were used for hemoglobin (Hb) typing. Amplification refractory mutation system (ARMS), restriction fragment length polymorphism (RFLP), gap-polymerase chain reaction (gap-PCR) and DNA sequencing analysis methods were used to determine genomic features. In this study, we found that 36 subjects carried homozygous mutations [IVS-I-110 (G>A) (HBB: c.93-21G>A) (58.3%), codon 8 (-AA) (HBB: c.25_26delAA) (5.6%), -30 (T>A) (HBB: c.-80T>A) (5.6%), IVS-I-6 (T>C) (HBB: c.92+6T>C) (5.6%) and IVS-II-1 (G>A) (HBB: c.315+1G>A) (5.6%)]. We found that 13 subjects carried compound heterozygosities for IVS-I-110/IVS-I-6 (15.4%) and IVS-I-110/frameshift codon (FSC) 44 (-C) (HBB: c.135delC) (15.4%). We observed that the Syrian subject also carried a compound heterozygosity for IVS-I-6/IVS-I-25 (-25 bp) (HBB: c.93_21del). We determined that the most frequently observed ß-thal mutation in the Çukurova region, where various types of hemoglobinopathies have been observed, is the IVS-I-110 mutation. As the prevalence of the disease will affect the region where the immigrant population is dense, population screening and prenatal diagnosis (PND) should be increased and the public should be made aware of the consequences.

Hemoglobinopathies in the Çukurova Region and Neighboring Provinces.

To contribute to the creation of a mutation map of the region, we aimed to determine the mutation spectrum of thalassemias and abnormal hemoglobins (Hbs) in the Çukurova region and surrounding provinces. In this study, a total of 8135 samples from Adana, Hatay, Mersin, Konya and Kayseri provinces between 1993 and 2014 were analyzed. Complete blood cell (CBC) counts and Hb typing were carried out using automatic cell counters, cellulose acetate membrane electrophoresis and high performance liquid chromatography (HPLC), respectively. For the molecular analyses, genomic DNA was extracted using both manual and automated DNA extraction devices. Determination of Hb mutations were done by microarray, restriction fragment length polymorphism (RFLP), amplification refractory mutation system (ARMS) and gap-polymerase chain reaction (gap-PCR) methodologies. Samples were analyzed for abnormal Hb and thalassemia mutations. Out of 8135 samples, 1382 were observed to be carrying Hb mutations. It was identified that 826 mutation carriers included abnormal Hbs with a frequency of 59.7%, 416 carriers included ß-thalassemia (ß-thal) mutations with a frequency of 30.7% and 136 carriers included α-thalassemia (α-thal) mutations with a frequency of 9.9%. In this study, the most frequently observed abnormal Hb in the region was Hb S [ß6(A3)Glu→Val (GTG > GAG), HBB: c.20T > A], whereas the most commonly observed mutations were the IVS-I-110 (G > A) (HBB: c.93-21G > A) point mutation in ß-thal and the 3.7 kb deletion in α-thal.

Glucose-6-phosphate dehydrogenase activity, structure, molecular characteristics and role in neonatal hyperbilirubinemia in cord blood in Cukurova region.

The most common causes of neonatal indirect hyperbilirubinemia are blood incompatibility and erythrocyte enzyme defects. Glucose-6-phosphate dehydrogenase (G6PD) is a guarantee of erythrocyte stability and capability of existence of red cells. We present here the results of a study on the effect of enzyme kinetics and different mutations on neonatal hyperbilirubinemia in the Cukurova region. Two hundred healthy term male neonates born in Cukurova University Balcall Hospital, Adana Maternity Hospital and Cukurova Maternal and Children's Hospital between 1 November 2004 and 30 November 2007 were consecutively studied. Nanogen DNA microarray was used to determine Gd Union, Gd San, Gd Mediterranean, and Gd San Antonio mutations. Quantitative G6PD enzyme assays were performed. Glucose-6-phosphate dehydrogenase deficiency was detected in six out of 200 male neonates (3%). The other 194 neonates had normal G6PD activity, with a mean of 8.3 +/- 2.1 IU/g hemoglobin (Hb) (5.2-12.7 IU/g Hb). Clinical follow-up, enzyme kinetics and genetic studies were performed in the G6PD-deficient neonates. Differences were observed in clinical outcomes, rates of bilirubin decline and maximum total bilirubin levels in the neonates having the same mutation. These differences might be caused by the effects of kinetic variant on the hyperbilirubinemia without the direct effect of the mutation. In future studies, mutation analyses of further G6PD-deficient cases may address the genotype differences and their clinical effects in G6PD-deficient patients.

Prenatal diagnosis of sickle cell anemia and beta-thalassemia in southern Turkey.

Hemoglobinopathies are the most common genetic diseases in Turkey. The incidence of sickle cell trait is 10.0% and beta-thalassemia (beta-thal) trait is 3.7% in the Cukurova region of southern Turkey. Sickle cell anemia is prevalent in the Cukurova region, but beta-thal is seen all over the country. A prenatal diagnosis center was established in 1992 at Adana, Turkey, for the prevention of sickle cell anemia and beta-thal. Fifteen hundred and seventy-five fetuses were examined at the Cukurova University Hospital, Adana, Turkey. Three hundred and eighty-six fetuses were diagnosed as homozygous or compound heterozygous for sickle cell anemia and beta-thal. A total of 15 different beta-thal mutations were characterized in the parents. The incidence of the IVS-I-110 (G > A) mutation accounted for about 50.0% of the parents with beta-thal trait. Twenty-four different genotypes were observed in this study. A total of 286 fetuses were diagnosed with homozygous sickle cell disease, 57 fetuses were beta-thal homozygotes, 25 fetuses were compound heterozygotes with Hb S [beta6(A3)Glu-->Val, GAG > GTG], and 18 of the fetuses were double heterozygotes for beta-thal mutations.

Beta globin gene cluster haplotypes of abnormal hemoglobins observed in Turkey.

Since the first observation of hemoglobin S (Hb S) in Turkey by Aksoy, the number of hemoglobin variants reported was increased. Beta globin gene cluster haplotypes are being used to determine the origin of the mutations under interest. We studied the beta globin gene cluster haplotypes for the six different abnormal hemoglobins which are Hb S, Hb D-Los Angeles, Hb G-Coushatta, Hb E, Hb E-Saskatoon and Hb J-Iran observed in Turkey. In this study, we report two novel haplotypes [- + + - - - +] and [- + - + + +] in association with the Hb G-Coushatta mutation. The haplotype for the Hb J-Iran is also reported as [- + - + + + +] for the first time in world populations from Turkey.

MyD 88 Polymorphisms in Children Diagnosed with Sepsis.

BACKGROUND: Myeloid differentiation primary response gene 88 (MyD 88) is an intracellular adapter protein that mediates the early immune response to pathogens. Toll-like receptors (except TLR-3) induce the immune response through a MyD 88-dependent signal pathway. AIMS: We aimed to investigate the MyD 88 polymorphisms that play important roles in the immune response in septic children and to evaluate whether or not they were risk factors in the development of sepsis. STUDY DESIGN: Case-control study. METHODS: Sixty-five patients diagnosed with sepsis in the Pediatric Intensive Care Unit during the period from April 2010 to January 2012 were included as the study group. Sixty-five children without sepsis were included as controls. After DNA was obtained from blood samples in the study and control groups, MyD 88 polymorphisms were analyzed. According to the genotype and allele frequencies, the distributions of MyD 88 polymorphisms [Single nucleotide polymorphism (SNP) - 938 C/A (rs4988453), MyD 88 SNP 1944 C/G (rs4988457)] were analyzed in both the study and control groups. RESULTS: The C/C genotype of MyD 88 SNP -938 was significantly more common than the C/A genotype in the patient group (p=0.002). No statistically significant difference in the frequency of the MyD 88 SNP 1944 genotype was found between the study and control groups (p=0.272). CONCLUSION: Gene polymorphism studies could elucidate our understanding of sepsis in terms of prevalence and the management of treatment. It was shown in this study that children with the MyD 88 SNP -938 C/C genotype had a greater tendency toward sepsis. However, additional studies should be performed.

Prenatal diagnosis of sickle cell anemia in twin pregnancies and identification by VNTRs.

BACKGROUND: Sickle cell anemia (SCA) is an inherited disorder of hemoglobin synthesis that is characterized by life-long severe hemolytic anemia, attacks of pain crisis, and chronic organ system damage. In this study, prenatal diagnosis was performed to three couples (families A, B, and C) with twin pregnancies who were at risk for SCA. METHODS: The SCA carrier state of the couples were confirmed at molecular level. Chorionic villus samples (CVS) of twins were obtained at 10-12 weeks of gestation. Amplification refractory mutation system (ARMS) and restriction fragment length polymorphism (RFLP) techniques were applied to determine and confirm the presence of the sickle cell anemia of the fetuses. Identification of each twin, confirmation of parentage and elimination of maternal contamination of chorionic villus samples were ruled out by variable number of tandem repeats (VNTR) analysis of four different loci [D1S80 (pMCT118), ApoB, IgJH, D4S95]. RESULTS: We found that one of the fetuses was heterozygous for SCA and the other was normal in family A; in family B both fetuses were heterozygous and in family C both fetuses were normal. CONCLUSION: Prenatal diagnosis is the major way of prevention of the genetic disorders including sickle cell anemia. Prenatal diagnosis of twins contains additional risks such as confusion in differentiation leading to false molecular diagnosis. VNTR loci analysis is a useful tool and can be safely used for the elimination of all problems mentioned above.

An Education Programme on Sickle Cell Anemia and ß-Thalassemia for the 8th Grade Students.

ß-thalassemia and sickle cell anemia (SCA) are major health problems in Çukurova. Screening studies have been carried on in this region for many years. The government started premarital screening programme and prenatal diagnosis has been done in Çukurova University Medical Faculty since 1992. In spite of all these, the prevention programme has not been successful. The purpose of this study was to start an education programme to increase the awareness of the people for these severe hereditary diseases. The target population was the 8th grade students. A visual presentation was given to students on Thalassemia and SCA after examining the curriculum in the Biology and/or the Science books. A total of 1221 students in seven Elementary Schools were informed. A questionnaire was given to five schools before the presentation and to two after the presentation. The comparison of the two group's result showed that the information given was well received. Thus, an extensive education programme encompassing doctors, health personel, civil and religious leaders and the support of the government will result in nil SCA and Thalassemia births.

Effect of oxidative stress on membrane proteins in thalassemia and iron deficiency anemia.

OBJECTIVE: To evaluate the effect of oxidative stress on membrane proteins according to relationship between malondialdehyde (MDA) levels and membrane protein ratios in different two patient groups. METHODS: Fifty-seven healthy control cases that had no symptoms of inherited blood diseases, 50 diagnosed ß-thalassemic patients, and 30 patients with iron deficiency anemia were enrolled. Erythrocyte membrane samples were prepared according to Dodge method and proteins were separated using 8.3% SDS-PAGE. Gels were stained with Coomassie Brillant Blue, then fraction quantities were determined by dansitometric method. MDA levels were measured by thiobarbituric acid method in plasma. One-way ANOVA test was used to compare parameters between groups. RESULTS: Significant difference in protein deficiency rates for spectrin and band 4.2 in group I, band 6 in group II and band 3 in group I and II when compared with control group (group III) were found. The significant difference between group I and II wasn't determined in spite of the fact that MDA levels of patients were different from the control group statistically. CONCLUSIONS: Any significant relationship statistically was not found. This consequence may be due to priority removal of oxidatively injured cells from the circulation in thalassemic patients and therefore, significant increase wasn't determined in the concentration of MDA.

Detection of 1311 polymorphism in the glucose-6-phosphate dehydrogenase gene by microarray technique.

INTRODUCTION: Nucleotide 1311 polymorphism at exon 11 of the glucose-6-phosphate dehydrogenase (G6PD) gene is fairly common in various populations worldwide, especially among Mediterranean populations. In this study, 1311 polymorphism in G6PD-deficient cases was identified by microarray technique. MATERIAL AND METHODS: Four hundred and fifty clinically healthy subjects were screened and 32 cases were found to have G6PD deficiency (7.11%). Our analysis of genomic DNA samples from 32 G6PD-deficient individuals revealed that the number and percentage of subjects who had a C-to-T alteration at nucleotide 1311 were 21 and 4.7% respectively. Given that the frequency of 1311 polymorphism has been reported in previous studies to be fairly high among G6PD-deficient people with the Mediterranean mutation, our data seem to be inconsistent with what we would expect for this particular region. RESULTS: The highly diverse ethnic background of the Adana population which probably results from the high level of immigration into this part of Turkey may be one of the most sensible explanations for this unexpected finding. Nevertheless, it seems that our results need to be confirmed in larger studies. CONCLUSIONS: The polymorphism studies in the G6PD gene may help us to illuminate the genetic basis of the G6PD deficiency in different regions and in various ethnic groups, and also to discover the influence of a specific polymorphism on the clinical course of the deficiency.

Vitamin D ameliorates stress ligand expression elicited by free fatty acids in the hepatic stellate cell line LX-2.

BACKGROUND/AIMS: Hepatic stellate cells play an important role as the major source of fibrillar and non-fibrillar matrix proteins in the process of liver fibrosis. Natural killer cells have an anti-fibrotic effect through the killing of activated hepatic stellate cells. Major histocompatibility complex class I-related molecules, MICA and MICB, function as ligands for the NKG2D receptor and play an important role in hepatic stellate cells susceptibility to natural killer cells during hepatic inflammation. The aim of this study was therefore to investigate the effect of vitamin D2 and free fatty acids on stress ligands and pro-fibrotic activity in LX-2 cells and human primary hepatic stellate cells. METHODS: LX-2 cells and primary human hepatic stellate cells were treated with vitamin D2 (10-6 M) and free fatty acids at different concentrations (0.25 mM, 0.5 mM, and 1 mM) for 24 hours, and expressions of the stress ligands MICA/B as well as of transforming growth factor-ß, α-smooth muscle actin and collagen 1α were assessed by quantitative real time-polymerase chain reaction. RESULTS: Treatment of cells with 0.5 mM and 1 mM free fatty acids induced α-smooth muscle actin and transforming growth factor-ß expression in LX-2 cells. Moreover, 1 mM free fatty acids resulted in increased expression of MICA. Surprisingly, collagen 1α expression was reduced after addition of free fatty acids. MICA/B expression in primary hepatic stellate cells was not affected by free fatty acids treatment. Vitamin D2 treatment significantly downregulated the free fatty acids-induced expression of transforming growth factor-ß and α-smooth muscle actin in LX-2 cells. Further, in hepatic stellate cells, a significant decrease in MICA/B mRNA with vitamin D2, independent of free fatty acids treatment, was detectable. CONCLUSIONS: These results indicate that vitamin D2 may reduce inflammatory and pro-fibrogenic activity of stellate cells in vitro.

Allele frequency distributions of Apo B VNTR locus in Cukurova, Turkey.

The highly polymorphic minisatellites contain a variable number of tandemly repeated (VNTR) DNA sequences. They are extremely useful and informative markers to study genetic variation among human populations. We have analysed the allele frequency distribution at the highly polymorphic apolipoprotein B (Apo B) VNTR locus in order to obtain the population data for the Cukurova region in Turkey by using the polymerase chain reaction and polyacrylamide gel electrophoresis. We observed 10 different alleles and 21 genotypes in a sample of 100 unrelated individuals. The allele frequencies ranged from 0.01 to 0.4, with an expected heterozygosity of 0.69 for the Apo B locus. Alleles 37 (frequency = 0.4) and 35 (frequency = 0.17) were the most common in the Cukurova population. There was a significant deviation from the Hardy-Weinberg equilibrium (HWE) for genotype frequencies (chi2 = 29.12; df = 1; p = 0.000). This study possesses novelty as it is the first DNA polymorphism study conducted at the Cukurova population using an Apo B minisatellite locus.