Investigation of the effects of Toxoplasma gondii on behavioral and molecular mechanism in bradyzoite stage.

Toxoplasma gondii is a single-celled parasite that causes a disease called toxoplasmosis. It can reach the central nervous system, but the mechanism of T. gondii disrupting the functioning of these brain regions occurs in bradyzoite stage of parasite, causing brain damage by forming tissue cysts in brain. In our study, the effects of T. gondii on locomotor activity, anxiety, learning and memory, and norepinephrine (NE), levodopa (L-DOPA), dopamine (DA) and 3,4-D-dihydroxyphenylacetic acid (DOPAC) catecholamines in amygdala, striatum, prefrontal cortex and hippocampus regions of the brain were investigated in bradyzoite stage. Twenty male Albino mice Mus musculus, 4-5 weeks old, weighing 20-25 g, were used. T. gondii inoculated to mice intraperitonealy with 48-50-hour passages of T. gondii RH Ankara strain. For intraperitoneal inoculation of mice 5x104 tachyzoites per mouse. No inoculation was made in control group (n: 20). Locomotor activity behavior in open field test (OFT), anxious behavior in elevated plus maze (EPM), and learning behavior in novel object recognition (NOR) tests were evaluated. NE, L-DOPA, DA and DOPAC were measured by HPLC in brain tissues of amygdala, striatum, prefrontal cortex and hippocampus. A decrease was observed in the locomotor activity, anxiety and learning values of the T. gondii group compared to the control group (p < 0.05). The heighten in NE and L-DOPA levels in amygdala tissue of T. gondii group compared to control group, an elevation in NE, L-DOPA, DA and DOPAC levels in striatum tissue, and an increase in levels of NE in prefrontal cortex tissue were detected in monoamine results. In hippocampus tissue, an increase was observed in DA levels, while a decrease was observed in NE, L-DOPA and DOPAC levels. In our study, it has been shown that T. gondii in bradyzoite stage reduces locomotor activity, causes learning and memory impairment, and has anxiogenic effects.

Association of clinicopathological features with E-cadherin (CDH1) gene-160 C>A promoter polymorphism in Turkish colorectal cancer patients.

BACKGROUND AND AIM OF STUDY: The role of E-cadherin (CDH1) gene-160 C>A (rs16260) promoter polymorphism in colorectal cancer (CRC) still remains inconclusive. The aim of this study is to investigate the associations between the CDH1-160 C>A polymorphism with the susceptibility and clinicopathological development of CRC in the Turkish patients. To our knowledge, this is the first report examining the role of CDH1 polymorphism in Turkish CRC patients. MATERIALS AND METHODS: A total of 92 colorectal carcinoma cases (including 62 colon and 30 rectal cancer patients) and the corresponding adjacent normal tissues as controls were studied. The polymorphism was genotyped using polymerase chain reaction-restriction fragment length polymorphism analysis. Clinicopathological features including patient's age, gender, tumor stage, and tumor location (colon/rectum) were compared statistically with the polymorphism status. RESULTS: There was no significant difference in both genotype and allele frequencies of the CDH1 polymorphism between colorectal tumor cases and normal samples (P = 0.472 and 0.508, respectively). Furthermore, no significant associations were observed between the CDH1 polymorphism status and age, gender, tumor stage, and tumor location of the colorectal tumor cases (all P > 0.05). CONCLUSIONS: These results indicate that CDH1-160 C>A polymorphism does not contribute to the genetic susceptibility of CRC and the polymorphism may not be a direct effect on the progression of the disease in Turkish CRC patients.

Effect of magnetic field on in vitro seedling growth and shoot regeneration from cotyledon node explants of Lathyrus chrysanthus boiss.

The stimulatory effects on germination of seeds and growth of plants of static magnetic field (MF) pre-treatments depending on MF intensity, exposure time periods, signal form, flux density, and source frequencies on plants are reported. Seed germination frequency is low due to dormancy in Lathyrus chrysanthus Boiss. from Fabaceae family, consisting of 187 taxa. Tissue culture protocol for this plant has already been optimized. This plant is also used as a model for developing alternative methods to overcome dormancy. This study was conducted to determine the effects of MF on in vitro seed germination, seedling growth, and shoot regeneration capacity of cotyledon node explants in Lathyrus chrysanthus Boiss. to obtain healthy seedlings in large quantities. The seeds of an ecotype (Diyarbakir) were subjected to 125 mT MF strength for different exposure time periods (0-untreated, 24, 48, and 72 h). Sterilized seeds were germinated on growth basal medium in Magenta vessels. Seed germination and seedling growth percentages were recorded after 7 and 14 days of culture initiation, whereas seedling and root lengths were noted 28 days after culture initiation. At the end of the culture, shoot regeneration percentage, shoot number per explant, highest shoot height per explant, and total shoot number per petri dish were recorded. According to the results, it could be concluded that MF treatment could clearly be used to improve germination by breaking dormancy not only in Lathyrus chrysanthus Boiss. but also other plant species. Bioelectromagnetics. 39:547-555, 2018.© 2018 Wiley Periodicals, Inc.

Assessment of markers expressed in human hair follicles according to different skin regions.

BACKGROUND: Body region-dependent hair follicle (HF) characteristics are concerned with follicular size and distribution, and have been demonstrated to have characteristics for each region of the body. OBJECTIVES: The aim of the present study was to investigate the expression patterns of the markers called cytokeratin 15 (K15), cytokeratin 6 (K6) and monoclonal antibody Ki-67, and also apoptosis in HFs, which can be observed in different parts of the human body. MATERIAL AND METHODS: In this study, healthy human HFs were taken by biopsy from 5 various donor sites of the human body: the scalp, the leg, the abdomen, the back and waist. HF-containing skin specimens taken using cryosection were stained with hematoxylin & eosin (H&E) and K15, K6, Ki-67 and terminal deoxynucleotidyl transferase-mediated digoxigenin-dNTP nick end-labelling (TUNEL) immunofluorescence staining protocol was performed. RESULTS: Different skin regions from the human body were examined histologically. While the HFs of scalp tissue showed anatomically obvious hair layers, some hair sections from other regions, like the leg, the abdomen, back and waist, were not as distinct as in the scalp region. According to our findings, K15 expression was highest in the scalp. In addition, the immunoreactivity (IR) intensity of K15 was significantly decreased in the HFs on the waist and abdominal regions, compared to the scalp and back regions (p < 0.001). However, the IR intensity of K6 in the scalp region was statistically significantly higher than the IR intensity of K6 in the abdomen region (p < 0.05). Moreover, we showed intraepithelial apoptosis and proliferation of keratinocytes in the bulge of HF. In the study, Ki-67-positive and TUNEL-positive cell numbers were not statistically significant (p > 0.05). CONCLUSIONS: Our findings are important for further investigation of molecular aspects of the human hair follicle stem cells compartments in health and disease, which might be a promising model for comparative studies with different human diseases.

Protective effects of curcumin and beta-carotene on cisplatin-induced cardiotoxicity: An experimental rat model.

OBJECTIVE: Cisplatin (CDDP) has been known to be an effective antineoplastic drug; however, it has a cardiotoxic effect. Curcumin (CMN) and beta-carotene (BC) have been suggested to protect biological systems against CDDP-induced damage. The current study was conducted to evaluate the possible protective roles of CMN and BC on CDDP-induced cardiotoxicity in rat cardiac tissues. METHODS: A total of 49 adult female Wistar albino rats were equally divided into seven groups as follows: control (no medication), sesame oil (1 mg/kg), CDDP (single dose injection two times as once a week, 5 mg/kg/week), BC (100 mg/kg), CDDP+BC (pretreated BC for 30 min before CDDP injection), CMN (200 mg/kg), and CDDP+CMN (pretreated CMN for 30 min before CDDP injection). These treatments were applied intraperitoneally for CDDP and with gavage for CMN and BC. The oxidative/antioxidant indicators, inflammatory cytokines, and histopathological alterations were examined. RESULTS: These alterations included a marked increase in malondialdehyde (MDA) level, significant decrease in catalase (CAT) and superoxide dismutase (SOD) activities, and significant elevation of tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, interleukin (IL)-6 in the CDDP group compared with the other groups. Histopathologically, CDDP-induced severe myocardial degenerative changes were observed. However, the CDDP-induced disturbances in the above-mentioned parameters significantly improved by treatment with BC and particularly CMN. CONCLUSION: This study indicated that CDDP treatment markedly caused cardiotoxicity; however, treatment with CMN or BC ameliorated this cardiotoxicity in rats. Furthermore, these findings revealed that treatment with CMN has a higher cardioprotective effect than that with BC against CDDP-induced cardiotoxicity in rat cardiac tissues.

Evaluation of Estrogen, Progesterone, and GCDFP-15 Expression in Uterine Leiomyomas.

Approximately, one fourth of women have leiomyomas. Leiomyomas are benign tumors that originated from smooth muscle cells. Estrogen is claimed to relate as a cause but exact mechanism has not fully understood. In this study, 95 leiomyoma cases that have been diagnosed by our department in years between 2010 and 2012 were examined. Age ranges of patients, sizes, locations, and numbers of leiomyomas were identified. Immunohistochemically ER (estrogen), PR (progesterone), and Gross cystic disease fluid protein-15 stains were performed to the paraffin blocks and their percentages of staining were noted. Statistically, submucosal and intramural locations were significantly related to ER and PR (p < 0,001). There were significant relationship between ER and PR in 30-50 years age group (p < 0,001). There were significant relationship between ER, PR and locations (p < 0,001), numbers (p < 0,001), sizes of leiomyomas (below 5 cm; p < 0,001), (between 5 and 10 cm; p = 0,037), larger than 10 cm; p = 0,002). Consequently, relationship between leiomyoma and ER, PR were revealed in this study. Also, relationship between leiomyoma locations and patient ages were identified statistically. There was no immunoreactivity with GCDFP-15 in leiomyomas.

Relationship of CD95 and COX-2 in renal cell carcinomas with survival and other prognostic parameters: A tissue microarray study.

OBJECTIVE: To investigate the expression of cyclooxygenase-2 and cluster of differentiation 95 in renal cell carcinomas having different clinico-pathological characteristics. METHODS: The study entailed histopathological diagnoses carried out on paraffin blocks at the Department of Pathology of the Medical Hospital of Duzce University, Turkey, between 2005 and 2011. Immunohistochemical staining for cyclooxygenase-2 and cluster of differentiation 95was performed on tissue microarray using standard procedures. Each patient's age and gender as well as the tumour's grade, stage, diameter, ureteral surgical margins, vascular invasion, capsule invasion and subtype were assessed. In order to determine if the cases were still alive, relatives were telephoned and identity registration records were checked. SPSS 18 was used for statistical analysis. RESULTS: There were 49 paraffin blocks in the study.Significant correlations were found between cyclooxygenase-2 and tumour subtype (p=0.044) as well as between cyclooxygenase-2 and tumour diameter (p=0.026). There was a significant correlation between cluster of differentiation 95and the Fuhrman grade (p=0.050). CONCLUSIONS: Expression of cluster of differentiation 95and cyclooxygenase-2 may be correlated with prognostic parameters in renal cell carcinoma and may also be associated with tumour progression.

High ionic strength depresses muscle contractility by decreasing both force per cross-bridge and the number of strongly attached cross-bridges.

An increase in ionic strength (IS) lowers Ca(2+) activated tension in muscle fibres, however, its molecular mechanism is not well understood. In this study, we used single rabbit psoas fibres to perform sinusoidal analyses. During Ca(2+) activation, the effects of ligands (ATP, Pi, and ADP) at IS ranging 150-300 mM were studied on three rate constants to characterize elementary steps of the cross-bridge cycle. The IS effects were studied because a change in IS modifies the inter- and intra-molecular interactions, hence they may shed light on the molecular mechanisms of force generation. Both the ATP binding affinity (K1) and the ADP binding affinity (K 0) increased to 2-3x, and the Pi binding affinity (K5) decreased to 1/2, when IS was raised from 150 to 300 mM. The effect on ATP/ADP can be explained by stereospecific and hydrophobic interaction, and the effect on Pi can be explained by the electrostatic interaction with myosin. The increase in IS increased cross-bridge detachment steps (k2 and k-4), indicating that electrostatic repulsion promotes these steps. However, IS did not affect attachment steps (k-2 and k4). Consequently, the equilibrium constant of the detachment step (K2) increased by ~100%, and the force generation step (K4) decreased by ~30%. These effects together diminished the number of force-generating cross-bridges by 11%. Force/cross-bridge (T56) decreased by 26%, which correlates well with a decrease in the Debye length that limits the ionic atmosphere where ionic interactions take place. We conclude that the major effect of IS is a decrease in force/cross-bridge, but a decrease in the number of force generating cross-bridge also takes place. The stiffness during rigor induction did not change with IS, demonstrating that in-series compliance is not much affected by IS.

Is the neutrophil-to-lymphocyte ratio indicative of inflammatory state in patients with obesity and metabolic syndrome?

OBJECTIVE: Obesity causes subclinical inflammation. Leukocyte count and high-sensitivity C-reactive protein (hs-CRP) are used to indicate inflammation in clinical practice. Also, inflammatory markers are evaluated as important indicators of cardiovascular risk in patients with obesity and metabolic syndrome (MetS). We aimed to investigate the usage of the neutrophil-lymphocyte ratio (NLR) as an inflammatory marker in obese patients with and without MetS. METHODS: The study included a total of 1267 patients. The patients were assigned groups according to degree of obesity and status of MetS. Metabolic and inflammatory markers were compared between groups, and correlation analysis was performed. RESULTS: Leukocyte count and hs-CRP were significantly different (p<0.001), but NLR was not different between body mass index (BMI) groups (p=0.168). Both lymphocyte and neutrophil counts were significantly increased with increased degree of obesity (p<0.001, p=0.028, respectively). Leukocyte, neutrophil, and lymphocyte counts and hs-CRP level showed a significant correlation with BMI (r=0.198, p<0.001; r=0.163, p<0.001; r=0.167, p<0.001; r=0.445, p<0.001, respectively), whereas NLR was not correlated with BMI (r=0.017, p=0.737). Only a significant association between a MetS severity of 5 and 4 with hs-CRP level was observed (p=0.028), whereas there was no statistically significant association for leukocyte count and NLR (p=0.246; p=0.643, respectively). CONCLUSION: NLR was not a good indicator of inflammation, while leukocyte and hs-CRP were more useful biomarkers to indicate inflammation in non-diabetic patients with obesity and MetS.

Does the MTHFR C677T gene polymorphism indicate cardiovascular disease risk in type 2 diabetes mellitus patients?

OBJECTIVE: Diabetes mellitus is a major risk factor for cardiovascular disease (CVD). We investigated the relationship among biochemical and cardiac risk parameters with the methylenetetrahydrofolate reductase (MTHFR) C677T genotype in type 2 diabetes mellitus (T2DM) patients. METHODS: One hundred seven T2DM subjects with severe CVD diagnosed by angiography were included consecutively in this cross-sectional study. Biochemical and clinical parameters were obtained from patients who were not positive for nephropathy and retinopathy. MTHFR C677T genotypes were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods. Normally and abnormally distributed continuous variables were analyzed using student t- and Mann-Whitney U tests. Categorical variables were analyzed using chi-square test. RESULTS: In the study, 31 T2DM subjects had the CC (29.0%), 62 had the CT (57.9%), and 14 had the TT (13.1%) genotypes. There were no significant differences between subjects with wild-type (677CC) and with mutant (677CT+677TT) alleles in terms of diabetes duration, visceral fat area, total cholesterol, triglyceride, fasting plasma glucose, systolic blood pressure, diastolic blood pressure, high-sensitivity C-reactive protein, homocysteine (Hcy), and carotid intima-media thickness values. CONCLUSION: This study suggests that MTHFR gene polymorphisms can not be used as a marker for the assessment of cardiovascular risk in T2DM patients.

Association of anxiety, sleepiness, and sexual dysfunction with restless legs syndrome in hemodialysis patients.

Restless legs syndrome (RLS) is characterized by unpleasant sensations, pain in the legs along with irresistible urges to move the legs when at rest. It is often accompanied by sleep disturbance. The purpose of this study was to assess the association of anxiety and sleepiness with sexual function in hemodialysis patients with and without RLS. Sociodemographic parameters, laboratory data of hemodialysis patients from three dialysis centers were collected prospectively. Anxiety, sleepiness, sexual function, and presence of RLS symptoms were assessed with standardized questionnaires as the RLS Diagnosis and Scale, Hamilton Anxiety Rating Scale, Epworth Sleepiness Scale (ESS), Arizona Sex Experiences Scale (ASEX). Univariate, regression tree method were used for statistical analysis. RLS was observed in 45.9% (n = 113) of hemodialysis patients (n = 246). The mean age of patients and duration of hemodialysis were 59.7 ± 14.0 and 4.9 ± 4.2 years, respectively. The correlation between Arizona Sexual Experiences Scale (ASEX) and sociodemographic features was significant (P < 0.0001). Patients with RLS had higher scores for anxiety (9.4 ± 7.8 with RLS and 6.8 ± 6.0 without), higher ESS (ESS, 6.6 ± 5.2 with RLS and 4.6 ± 4.0 without), and higher ASEX (24.6 ± 5.7 with RLS and 22.5 ± 6.8 without) than did those without RLS. The presence of RLS symptoms in hemodialysis patients was associated with sleepiness, anxiety, and sexual dysfunction. A regression tree method, which is a different statistical method, can help physicians estimate patients ASEX, RLS, ESS, and anxiety scores.

Association of endothelial nitric oxide synthase gene polymorphisms (894G/T, -786T/C, G10T) and clinical findings in patients with migraine.

Migraine is a common neurological disorder characterized by recurrent attacks, unilateral head pain, and related symptoms. The aim of this study was to investigate three endothelial nitric oxide synthase (eNOS) polymorphisms in 176 patients with migraine and 123 healthy individuals. Clinical and biochemical parameters were investigated. Genetic analysis was performed using the polymerase chain reaction-restriction fragment length polymorphism method. The differences between migraine cases and the control group were significant for two polymorphisms (-786T/C and 894G/T) (p = 0.000). Homocysteine and body mass index (BMI) were significantly higher in the migraine group than in the control group (p = 0.001 and p = 0.000). The relation between -786T/C genotype and BMI and allodynia was significant. TC heterozygotes and CC homozygotes were significantly higher in the migraine group than in the control group (OR 2.843 and 95 % CI 1.681-4.808 and OR 3.729 and 95 % CI 1.784-7.792, respectively). The 894G/T genotype was correlated with BMI, pain intensity, age at the onset of migraine, nausea, tension, compression, and allodynia. For this polymorphism, GT heterozygotes and TT homozygotes were significantly higher in the migraine group than in the control group (OR 3.027 and 95 % CI 1.830-5.008 and OR 3.221 and 95 % CI 1.223-8.484, respectively). The G10T genotype was correlated with attack duration and age at the onset of migraine (p = 0.008 and p = 0.040). eNOS polymorphisms may be useful markers for assessing migraine risk and clinical diagnosis.

Endothelial nitric oxide synthase gene polymorphisms (promoter -786T/C, exon 894 G/T and intron G10T) in unexplained female infertility.

BACKGROUND/AIMS: Recent investigations in both males and females show that there may also be some genetic risk factors associated with infertility, and endothelial nitric oxide synthase (eNOS) has important functions in implantation. We aimed to investigate the association of three different polymorphisms of eNOS (promoter -786T/C, exon 894 G/T and intron G10T) with unexplained female infertility. MATERIALS AND METHODS: Two groups of patients were included in the study: (1) women with unexplained infertility and (2) healthy, fertile women with normal menstrual cycles. eNOS polymorphisms were studied in genomic DNA of each patient by polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: Forty-one women with unexplained infertility and 40 fertile women were included. Baseline physical characteristics and hormonal parameters of the two groups were similar. For eNOS exon 894 G/T polymorphism, the GG homozygotes were significantly lower and the heterozygotes GT were significantly higher in the infertile group than in the control group (p < 0.05). eNOS gene polymorphism both for promoter and intron were similar in the two groups (p > 0.05). CONCLUSION: Altered eNOS protein caused by eNOS exon 894 G/T polymorphism might cause implantation failure, which may be a possible cause of unexplained female infertility.

Omentin serum levels and omentin gene Val109Asp polymorphism in patients with psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory disease of uncertain pathogenesis. Omentin is a new adipokine with anti-inflammatory properties; however, the relationship between psoriasis and omentin has not been fully established yet. OBJECTIVES: This study was designed to evaluate the relationship between psoriasis and omentin serum levels and Val109Asp polymorphism in exon 4 of the omentin gene. METHODS: Forty-nine patients with plaque-type psoriasis and 39 healthy subjects were included in the study. Omentin concentrations were determined by using enzyme-linked immunosorbent assay. Val109Asp polymorphism in exon 4 of the omentin gene was assessed by the polymerase chain reaction-restriction fragment length polymorphism method. Genotypes were determined according to the bands formed in agarose electrophoresis gels. In the statistical analysis, the level of significance was set at P < 0.05. RESULTS: The serum omentin levels of the patients with psoriasis (354.2 ± 152.0) were found to be significantly lower than those in the control group (488.7 ± 190.3) (P = 0.001). A moderate level negative correlation was determined between serum omentin level and body mass index and waist circumference. No significant differences were observed between the patient and control groups in terms of the genotype and allele frequency of Val109Asp polymorphism in exon 4 of the omentin gene (P > 0.05). CONCLUSIONS: Omentin serum levels were determined to be low in patients with psoriasis. No significant difference was found regarding Val109Asp polymorphism of the omentin gene. To the best of our knowledge, our study is the first clinical study to examine the relationship between psoriasis and omentin in terms of serum and genomic levels.

Investigation of MTHFR C677T gene polymorphism, biochemical and clinical parameters in Turkish migraine patients: association with allodynia and fatigue.

We investigated whether there is any relationship between biochemical and clinical parameters of migraine and methylenetetrahydrofolate reductase (MTHFR) gene C677T polymorphism, associated with the migraine subtypes, symptoms, and gender. A total of 150 migraine patients with and without aura (MA and MO) and 107 non-sufferers were included in the study. Biochemical and clinical parameters were measured and genetic analysis was performed. The MTFHR C677T genotype was significantly higher in the migraine group (p = 0.000). The CT genotype frequency of individuals with a family history of migraine was significantly higher (p = 0.025). This genotype frequency was higher in patients who suffer from compression, allodynia, fatigue, and sleeplessness (p = 0.027, 0.023, 0.006, and 0.05, respectively). Homocysteine and total cholesterol levels were significantly higher in the migraine group than the control group (p = 0.007 and 0.010, respectively). However, the other biochemical and clinical parameters did not differ from each other (p > 0.05), with only attack frequency being significantly higher in the MO group (p = 0.005). While the folate and HDL levels were significantly higher in females (p = 0.001 and 0.000, respectively), the homocysteine and triglyceride levels were significantly higher in males (p = 0.000 for each one). BMIs were significantly lower in the control than the migraine group (p = 0.021); however, an association between the C677T variant and BMI was not found (p = 0.787) in the migraine group. An association between the MTHFR C667T polymorphism and migraine susceptibility was found. Additional studies including genetic, clinic, and biochemical parameters should be conducted to better understand the disease.

First observation of Hb Tunis [beta124(H2)Pro>Ser] in Turkey.

Hb Tunis [beta124(H2)Pro>Ser] was reported from Tunisia in 1988. This hemoglobin variant was detected by isoelectric focusing moving just ahead of Hb A. It cannot be identified by standard hemoglobin electrophoresis due to its similar mobility to Hb A. It has normal stability and oxygen affinity and does not produce any clinical symptoms. Here, we report a heterozygous Hb Tunis [beta124(H2)Pro>Ser] case discovered for the first time in Turkey in a premarital screening program. This hemoglobin variant can be identified with high performance liquid chromatography analysis confirmed with DNA sequencing. We emphasize in our study the importance of an interdisciplinary collaborative study at the provincial basis for the success of the hemoglobinopathy control program.

Hb D-Los Angeles [beta121(GH4)Glu>Gln] and Hb Beograd [beta121(GH4)Glu>Val]: Implications for their laboratory diagnosis and genetic origins.

OBJECTIVE: The aim of this study was to determine the laboratory diagnosis and genetic origins of the hemoglobin (Hb) variants, Hb D-Los Angeles and Hb Beograd observed frequently in our region. MATERIAL AND METHODS: Hb variants were investigated in one Hb D-Los Angeles and two Hb Beograd families. These families were unrelated with each other. For the determination of Hb variants, alkaline/acid electrophoresis, HPLC, DE-52 micro-column chromatography procedures were applied. Mutations were determined by non-radioactive fluorescence automated DNA sequencing. Beta globin gene cluster haplotypes were identified by RFLP analysis at seven loci known as ε-Hinc II, Gγ-Hind III, AΨß-Hind III, 5'Ψß-Hinc II, 3'Ψß-Hinc II, ß-Ava II ve 3'ß-Hinf I. RESULTS: Three novel beta globin gene cluster haplotypes were identified as in relation with Hb D-Los Angeles [--+-+++], Hb Beograd [+----++ and -+-(+/-)(+/-)+(+/-)]. These haplotypes were reported for the first time in the world population Conclusion: In this study we emphasize the importance of DNA seqeuncing and other laboratory procedures for the identification of Hb variants in premarital diagnosis. On the other hand we discuss also the genetic origins of these Hb variants.

Beta globin gene cluster haplotypes of the beta thalassemia mutations observed in Denizli province of Turkey.

OBJECTIVE: Our aim is to identify the beta globin gene cluster haplotypes for the beta thalassemia mutations in Turkey at regional basis. Beta thalassemia mutations included in this study were IVS-I-110 (G>A), FSC 8/9 (+G), IVS-II-1 (G>A), IVS-I-5 (G>C), IVS-I-1 (G>A), IVS-I-6 (T>C) and FSC 8 (-AA). METHODS: We studied 22 unrelated patients with ß-thalassemia major and 72 unrelated healthy subjects from our Department's DNA bank. Haplotype analysis was done by polymerase chain reaction (PCR)-based restriction enzyme digestion for the beta globin gene cluster of the following polymorphic restriction sites: Hinc II 5' to ε, Hind III 5' to Gγ, Hind III in the IVS-II 5' to Aγ, Hinc II in pseudo ß, Hinc II 3' to pseudo ß, Ava II in ß, Hinf I 3' to ß. Associated haplotypes for the normal control samples (72 individuals, 144 chromosomes) were determined by Arlequin 3.1 software with unknown gametic phase. RESULTS: According to the results obtained, the most frequent beta globin gene cluster haplotypes in the normal population are (+----++), (+----+-), (-+-++++), (+-----+) with the frequencies of 28.6 %, 17.2 %, 9.8 % and 8.3 % respectively. IVS-I-110 mutation is linked with the haplotypes (+----++) and (+-----+). Observed haplotypes are (+----++) for FSC 8/9 (+G), (-+-+++-) for IVS-II-1 (G>A), (-+-++-+ and -+-++++) for IVS-I-5 (G>C), (+----+- and +------) for IVS-I-1 (G>A), (-++---+) for IVS-I-6 (T>C) and (+-----+) for FSC 8 (-AA). CONCLUSION: In conclusion, our region shows the Mediterranean character for the beta thalassemia mutations. According to the obtained results, IVS-I-110 (G>A) mutation linked with haplotype VII (+-----+), IVS-I-5 (G>C) mutation with haplotype IV (-+-++-+), codon 8/9 (+G) linked with haplotype I (+----++) were shown for the first time in Turkish population. The linkage of haplotype (+------) with the IVS-I-1 (G>A) mutation is reported for the first time in the published literature. In Denizli province of Turkey, beta globin gene cluster haplotypes of the normal population are strongly associated with the haplotypes of I (+----++), V (+----+-) and IX (-+-++++) respectively.

First observation of Hb D-Ouled Rabah [beta19(B1)Asn>Lys] in the Turkish population.

Hb D-Ouled Rabah [beta19(B1)Asn>Lys] is a rare hemoglobin (Hb) beta chain variant reported from Tuareg tribes in Algeria and once from China. It was suggested that Hb D-Ouled Rabah might be specific of Berber-speaking populations. Our report describes the first observation of this hemoglobin variant in the Turkish population.

Genetic origin of Hb D-Los Angeles [beta121(GH4)Glu-->Gln, GAA-->CAA] according to the beta-globin gene cluster haplotypes.

Hb D-Los Angeles (also known as D-Punjab, D-North Carolina, D-Portugal, D-Chicago and Oak Ridge) is an abnormal hemoglobin (Hb) with an amino acid substitution of glutamine for glutamic acid at codon 121 of the beta-globin gene. The origin and spread of Hb D-Los Angeles is not known. This is due to lack of information and remains to be elucidated. According to published reports, the Hb D-Los Angeles mutation is mostly linked with Mediterranean haplotype I [+ - - - - + +]. Besides the Mediterranean haplotype, a novel haplotype was also reported from Thailand [- - + + - - + + +]. Here we report a new haplotype from Turkey [- + -- + + +] that has not been described before. These results suggest that the Hb D-Los Angeles mutation has at least three different genetic origins.