Halil Gürhan Karabulut

Molecular alterations in the TP53 gene of peripheral blood cells of patients with chronic myeloid leukemia

The TP53 gene has been extensively studied in patients with chronic myeloid leukemia (CML), both in chronic phase and in blast crisis. Mutations in the gene were found in up to 30% of the patients, especially among those in blast crisis. We report the results of an analysis of 29 blood samples from CML patients: 8 samples from chronic phase patients, 8 from patients in the accelerated phase, and 13 from patients in blast crisis. By using genomic DNA, we sequenced PCR products of the coding exons and most introns of the TP53 gene, finding genetic changes in 30% of the blast crisis samples and 12% in chronic phase. All mutations were found in introns and were previously unreported. Immunocytochemical studies revealed accumulation of TP53 in blood cells of samples both from chronic phase and blast crisis patients. Since these samples had no TP53 mutations, we believe that wild type TP53 accumulates in blood cells of CML patients. Our results, therefore, indicate that molecular changes in coding regions of the TP53 gene are rare. The significance of the abundance of intronic changes should be investigated further. Accumulation of wild type TP53 in CML cells may indicate an additional mechanism involving this gene in the pathogenesis of this disease. Genes Chromosomes Cancer 21:2–7, 1998.

The Frequency of A1298C and C677T Polymorphisms of the Methylentetrahydrofolate Gene in Turkish Patients with Rheumatoid Arthritis: Relationship with Methotrexate Toxicity.

The C677T and A1298C polymorphisms of methylenetatrahydrofolate reductase (MTHFR) gene are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine the frequency of MTHFR C677 T and A1298C gene polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity. Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ±12.5 and 46.2 ± 13.4 years, were enrolled to the study. Demographic characteristics were obtained and MTX-related adverse effects were recorded in the patient group. The A1298C and C677T polymorphisms of the MTHFR gene were analyzed and the distribution of genotypes according side effects, were determined. The frequency of MTHFR C677T and A1298C polymorphisms were similar in the patient and control groups. Folic acid supplementation with a mean dose of 5mg folic acid/week, was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment, and MTX had been discontinued in 12 (18.8%) patients due to side effects and/or inefficacy. MTHFR C677T and A1298C gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. In conclusion, A1298C and C677T polymorphisms in the MTHFR gene, were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible from the MTX toxicity in patients suffering from RA.

Relation between the insertion/deletion polymorphism of the angiotensin I converting enzyme gene and restenosis after coronary stenting.

Background Observations with intravascular ultrasound demonstrated that neointimal hyperplasia is the predominant factor responsible for in-stent restenosis. Experimental data suggest that angiotensin I converting enzyme (ACE) plays a role in the thickening of neointima after balloon denudation. Insertion/deletion (I/D) polymorphism of the ACE gene is significantly associated with plasma level of ACE and subjects with D/D genotype have significantly higher plasma levels of ACE than normal. Objective To investigate whether this polymorphism influences the risk of restenosis after coronary stenting. Methods We genotyped 158 patients who had undergone single-vessel coronary stenting for the ACE I/D polymorphism. Results Of the 158 patients, 56 (35%) had the D/D genotype, 71 (45%) had the I/D genotype and 31(20%) had the I/I genotype. Prevalences of genotypes were compatible with Hardy-Weinberg equilibrium and distributions of ACE genotype among patients and 132 healthy controls from the same geographic area did not differ. At follow-up (after a median duration of 5.4 months), overall rates of angiographic restenosis and of revascularization of target lesion (RTL) were 32.3 and 22.8%, respectively. Of 51 patients with angiographic restenosis, 31 (60.8%) had focal and 20 (39.2%) had diffuse patterns of restenosis. Diffuse in-stent restenosis was significantly more prevalent among patients with D/D genotype (P= 0.016). Multiple stepwise logistic regression analysis identified ACE I/D polymorphism as the independent predictor of angiographic restenosis and RTL. Relative risk of angiographic restenosis was 6.29 [95% confidence interval (CI), 1.80–22.05, P= 0.0004] for D/D genotype and 3.88 (95% CI 1.11–13.12, P= 0.029) for I/D genotype, whereas relative risk of RTL was 7.44 (95% CI 1.60–34.58, P= 0.01) for D/D genotype and 3.88 (95% CI 0.083–18.15, P= 0.085) for I/D genotype. Conclusions The ACE I/D polymorphism is significantly associated with risk of angiographic and clinical restenosis after coronary stenting. Angiographic pattern of restenosis is also significantly associated with I/D polymorphism, diffuse type being more prevalent among subjects with D/D genotype.

WNT Signaling Perturbations Underlie the Genetic Heterogeneity of Robinow Syndrome

Locus heterogeneity characterizes a variety of skeletal dysplasias often due to interacting or overlapping signaling pathways. Robinow syndrome is a skeletal disorder historically refractory to molecular diagnosis, potentially stemming from substantial genetic heterogeneity. All current known pathogenic variants reside in genes within the noncanonical Wnt signaling pathway including ROR2, WNT5A, and more recently, DVL1 and DVL3. However, ∼70% of autosomal-dominant Robinow syndrome cases remain molecularly unsolved. To investigate this missing heritability, we recruited 21 families with at least one family member clinically diagnosed with Robinow or Robinow-like phenotypes and performed genetic and genomic studies. In total, four families with variants in FZD2 were identified as well as three individuals from two families with biallelic variants in NXN that co-segregate with the phenotype. Importantly, both FZD2 and NXN are relevant protein partners in the WNT5A interactome, supporting their role in skeletal development. In addition to confirming that clustered -1 frameshifting variants in DVL1 and DVL3 are the main contributors to dominant Robinow syndrome, we also found likely pathogenic variants in candidate genes GPC4 and RAC3, both linked to the Wnt signaling pathway. These data support an initial hypothesis that Robinow syndrome results from perturbation of the Wnt/PCP pathway, suggest specific relevant domains of the proteins involved, and reveal key contributors in this signaling cascade during human embryonic development. Contrary to the view that non-allelic genetic heterogeneity hampers gene discovery, this study demonstrates the utility of rare disease genomic studies to parse gene function in human developmental pathways.

The relationship between angiotensin converting enzyme gene I/D polymorphism and qt dispersion in patients with hypertrophic cardiomyopathy

Introduction: Hypertrophic cardiomyopathy (HCM) is characterized by disorganized myocardial architecture, and may cause ventricular arrhythmias and sudden death. The angiotensin-converting enzyme (ACE) with two deletion alleles (DD genotype) has been proposed to be associated with increased myocardial collagen content. We evaluated QT dispersion (QTd), which reflects regional differences in ventricular repolarization, in HCM patient and controls among the three different ACE genotypes. Materials and methods: Sixty-three patients with HCM and 20 healthy subjects were included in the study. QT parameters were measured from 12 lead electrocardiograms. ACE genotypes were determined from the DNA extracted from peripheral blood by a polymerase chain reaction (PCR) method. QT parameters were compared among the three ACE genotypes both in HCM patients and controls. Results: Median ages were similar in HCM and control groups. QTd and corrected QTd (QTcd) were significantly greater in the HCM group compared with the controls. The frequencies of each genotype were similar in both groups. Although QTd and QTcd did not differ among the three genotypes in the control subjects, they were significantly greater in patients with DD genotype compared with other genotypes in the HCM group. Conclusion: QTd and QTcd are increased in patients with HCM, especially in those with the DD genotype.

Relationship Between Angiotensin-Converting Enzyme Gene Polymorphism and Severity of Aortic Valve Calcification

OBJECTIVE To investigate the role of angiotensin-converting enzyme (ACE) gene polymorphism in patients with degenerative aortic valve calcification (AVC). PATIENTS AND METHODS Our study consisted of 305 Turkish patients of European descent (139 male, 166 female; mean plus or minus age, 68 plus or minus 9 years) referred to our echocardiography laboratory for aortic valve evaluation between June 2, 2003, and April 29, 2005. The severity of AVC was graded from 1 to 6 by echocardiography. We used polymerase chain reaction to determine ACE gene polymorphism. RESULTS The ACE insertion/deletion genotype distributions for the study population were in Hardy-Weinberg equilibrium (chi square equals 3.5, P equals .18). The study population was divided into 3 groups based on the severity of AVC: those with grade 1 calcification were in group 1, those with grades 2 to 4 in group 2, and those with grades 5 to 6 in group 3. Group 1 patients were significantly younger, less likely to have hypertension and diabetes, and had higher high-density lipoprotein cholesterol levels. The genotype frequencies were significantly different among groups, with the insertion/insertion genotype being less prevalent in group 3 patients. In multivariate analysis, independent predictors of severe AVC were hypertension (odds ratio [OR], 5.6; 95% confidence interval [CI], 2.8 to 11.0; P less than .001), low high-density lipoprotein cholesterol (OR, 2.7; 95 percent CI, 1.5 to 4.9; P equals .001), and the deletion/deletion and insertion/deletion vs insertion/insertion genotype (OR, 3.2; 95 percent CI, 1.5 to 7.2; P equals .004). CONCLUSION These results suggest that ACE gene polymorphism may be associated with severe AVC.

Polimorfismo do gene timidilato sintase e nível plasmático total de homocisteína em um grupo de pacientes turcos com artrite reumatoide: relação com a atividade da doença e toxicidade ao metotrexato

BACKGROUND The polymorphism of thymidylate synthase (TS) gene and homocysteine are reported to have a relationship to methotrexate (MTX) metabolism, with conflicting results. The aim of this study was to determine homocysteine levels and the frequency of TS gene triple repeat (TS3R) and double repeat (TS2R) polymorphisms in a group of Turkish RA patients and evaluate its association with MTX toxicity and disease activity. METHODS Sixty-four patients with RA and 31 control subjects with a mean age of 48.7 ± 12.5 and 46.2 ± 13.4 years, were enrolled to the study. Demographic characteristics were obtained and number of patients with MTX-related adverse affects, were recorded in the patient group. The homocysteine levels and TS2R/TS3R polymorphisms of the TS gene were analyzed and the distribution of genotypes according to MTX toxicity and disease activity, were determined. RESULTS The demographic properties were similar between the patient and control subjects. Folic acid supplementation with a mean dose of 5mg folic acid/week, was present in all patients. Thirty-six of the 64 patients showed adverse effects to MTX treatment. The frequency of TS2R and TS3R polymorphisms were found to be similar in the patient and control groups. TS2R and TS3R gene polymorphisms were found to be similar in patients with and without MTX-related adverse events. The mean homocysteine level was also similar in patients with and without TS gene polymorphism, but was found to be higher (12.45μmol/L vs 10.7μmol/L) in patients with MTX-related side effects than in patients without side effects. The mean level of homocysteine was correlated with levels of ESR in the patient group. CONCLUSIONS In conclusion, homocysteine levels might effect the disease activity and toxicity of MTX but 2R and 3R polymorphisms in the TS gene, were not related with MTX-related toxicity in RA patients receiving folate supplementation. Further studies are needed to illuminate the polymorphisms in other enzymes that might be responsible from the MTX toxicity in patients suffering from RA.

DNA methyltransferase expression differs with proliferation in childhood acute lymphoblastic leukemia

DNA methylation is involved in genomic imprinting, tissue and stage specific gene regulation, X chromosome inactivation and especially necessary in normal mammalian development [1]. Methylation of DNA at cytosine base occurs at most CpG islands in the mammalian genome, with exception of CpG islands of gene promoters which are usually unmethylated. Methylation of promoter CpG islands inhibits the activity of the gene by inhibiting the association of some DNA-binding factors with methylated DNA sequences or by silencing transcription via methyl-CpG binding proteins with co-repressors and chromatin modifiers [2]. Cancer is known as a genetic disease, in which epigenetic alterations play important roles. Cancer cells show global genomic hypomethylation, but the CpG islands of the promoters, especially the promoters of the tumour suppressor genes are hypermethylated [3]. Aberrant methylation of CpG islands is a major epigenetic mechanism of gene expression in human cancers. Global hypomethylation is important in tumour formation by different ways which include chromosomal and genomic instability, increased mutation rate and recombination frequency [4]. Hypermethylation of tumour suppressor genes causes gene inactivation, furthermore aberrant DNA methylation facilitates gene mutation, because deamination of 5-methylcytosine leads to the formation of thymine which is not repaired by uracil glycosylases, instead of the formation of uracil by cytosine deamination [5]. Acute lymphoblastic leukemia (ALL), is the most frequent cancer of childhood (25%). The blockage of lymhpoid cell development at any stage, leads to ALL. Chromosomal aberrations as well as epigenetic changes are important in it’s development. E-cadherin, DAP-kinase, p73 and p15 gene promoters are some of the gene promoters that are known to be hypermethylated in childhood ALL [6–8]. DNA methyltransferases (DNMT’s) are the enzymes that methylate DNA. In mammalians, there are five different DNMT’s: DNMT1, DNMT2, DNMT3A, DNMT3B and DNMT3L. All of the DNMT’s, except DNMT3L, are catalitically active. All have a catalytic carboxy terminal and a regulator amino terminal, except DNMT2. The activity of DNMTs are greatly regulated by their amino terminal domain which interacts with other molecules. DNMT2, contains only the catalytic domain and participates scarcely to the formation of DNA methylation pattern although it covalently binds to DNA [9–11]. DNMT1 targets the replication fork, methylates preferentially hemimethylated DNA, and is responsible for the Isik Bokesoy—Retired Professor.

Skin-Dominant Phenotype in a Patient with H Syndrome: Identification of a Novel Mutation in the SLC29A3 Gene.

H syndrome (OMIM 602782) is a very rare autosomal recessive genodermatosis with multisystem involvement. Hallmarks of this disorder are juvenile onset and progressive, hyperpigmented, hypertrichotic lesions with histiocytic infiltration. Associated systemic manifestations form a long list, and there is high variability between patients. In some patients, dysmorphic and other systemic features may be so subtle that the disorder may readily be mistaken as an acquired skin disease and treated as such. Herein, we report a novel homozygous c.1339G>A (p.Glu447Lys) mutation in the SLC29A3 gene in a patient with skin-dominant presentation of H syndrome. Additionally, due to the present case, double superior vena cava can be added to the list of possible cardiovascular manifestations of H syndrome.

Investigation of androgen receptor gene mutations in a series of 21 patients with 46,XY disorders of sex development.

Abstract Aim: Androgen receptor (AR) gene mutations are the leading cause of 46,XY disorders of sex development (DSD) and are associated with varying degrees of androgen insensitivity. The aim of this study is to investigate AR gene mutations in 46,XY DSD patients with normal testosterone secretion, either normal or high testosterone/dihydrotestosterone (T/DHT) ratio and normal SRD5A2 gene analysis, collectively, suggestive of androgen insensitivity syndrome (AIS). Methods: We direct sequenced all eight exons of the AR gene in 21 index patients with varying degrees of undervirilization. Results: We detected AR gene alterations in five patients. In patients with complete AIS we found p.Val30Met in exon 1 and p.Gly689* in exon 4. One patient with partial AIS had p.Gln712Glu in exon 4. In two patients with partial phenotype, we found common p.Glu213Glu (c.639G>A) SNP, and an additional p.Ile817Ile (c.2451T>C) mutation was found in one of these two patients. Discussion: Despite the fact that T/DHT ratio is frequently used in diagnosis of AIS, lack of precisely determined cutoffs compromises correct diagnosis. Hence, depending on clinical and biochemical findings solely may delay correct diagnosis. Direct sequence analysis of the AR is essential for precise diagnosis of AIS.