Fuat Dilmec

Investigation of CTLA‐4 and CD28 gene polymorphisms in a group of Turkish patients with colorectal cancer

Colorectal cancer (CRC), also called colon cancer or bowel cancer, includes cancerous growths in the colon, rectum and appendix. The immune system is an important defence mechanism against cancer and is often dysfunctional in patients with malignancies. Cytotoxic T lymphocyte‐associated antigen‐4 (CTLA‐4) and CD28 genes encode receptors that provide negative and positive signals, respectively. Polymorphisms in these genes can affect their functions. In this study, we aimed to investigate the association of cancer with the frequencies and roles of CTLA‐4/+49A > G (exon 1) and –318C > T (promoter), and CD28/IVS3 + 17T > C (intron 3 position + 17). These polymorphisms were genotyped using polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) in 218 Turkish subjects (56 patients with CRC and 162 healthy controls). No statistically significant differences in the genotype distributions of CTLA‐4/+49GG (1.8% vs. 6.8%, odds ratio (OR) = 0.250, P = 0.305) and CTLA‐4/−318TT (0% vs. 0.6%, OR = 1.006, P = 1.000), and CD28/IVS3 + 17CC (8.9% vs. 3.7%, OR = 0.2411, P = 0.155) between patients with CRC and healthy controls, were observed. We also found that there were no significant differences in the frequencies of CTLA‐4/+49G (18.8% vs. 20.1%, OR = 0.920, P = 0.891) and CTLA‐4/−318T (7.1% vs. 4.3%, OR = 1.653, P = 0.314), and CD28/IVS3 + 17C alleles (25.9% vs. 19.1%, OR = 1.353, P = 0.139) between two study groups. Present results suggested that CTLA‐4 and CD28 gene polymorphisms did not play an important role in Turkish patients with CRC.

Identification of Leishmania parasites in clinical samples obtained from cutaneous leishmaniasis patients using PCR-RFLP technique in endemic region, Sanliurfa province, in Turkey

Antroponotic cutaneous leishmaniasis (ACL) is an endemic disease and one of the major health problems in Sanliurfa province located in the southeastern region of Turkey. Leishmania tropica is confirmed as the causative agent of ACL in this region. In Sanliurfa city alone, the recorded total cases of ACL were 6,817 between 2001 and 2006. We aimed to determine the effectiveness of a polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) method for identification and differentiation of the Leishmania parasite in comparison to direct microscopic examination of clinical samples. The lesion exudates were collected from 51 ACL suspected patients and used for smear-slide preparations and DNA isolation. The isolated DNA was amplified by PCR, including primers selected on repetitive DNA for identification of a Leishmania subgenus, and the amplified DNA was restricted by HaeIII restriction endonuclease. The PCR-RFLP results showed that only L. tropica exists in this province. It is also determined that the positivity rate with PCR was higher (96%) than by microscopic examination (64%) in the diagnosis of ACL. Our results indicate that the PCR-RFLP method is more sensitive and specific for the detection and differentiation of agents of ACL in this area.

Monitoring of failure of chloroquine treatment for Plasmodium vivax using polymerase chain reaction in Sanliurfa province, Turkey

Malaria is a complex disease that varies widely in epidemiology and clinical manifestation in the southeastern part of Turkey. In many regions of the world, chloroquine (CQ) has been the standard treatment for Plasmodium vivax. However, the resistance of the Plasmodium species to antimalarial drugs has become an increasing problem and a concern worldwide. Our target was to determine the Plasmodium species in the southeast region of Turkey and the therapeutic efficacy of CQ used in the treatment of malaria. Blood samples were collected from 180 patients infected with malaria before and after CQ treatment and were subjected to DNA isolation. The isolated DNA was amplified by a seminested multiplex polymerase chain reaction (SnM-PCR) including primers selected on Plasmodium small subunit ribosomal RNA (ssrRNA) genes for identification of the malaria species. The SnM-PCR results showed that only P. vivax exists in this province. It was also determined that there is a therapeutic failure to CQ in 9.5% of patients. These were the second report on identification of P. vivax and the third report on determination of the therapeutic failure in patients who used CQ to cure human malaria in the southeastern region of Turkey. Our results demonstrate that the SnM-PCR is a sensitive, specific, and a rapid tool for the differentiation of malaria species.

Beta-globin gene mutations in children with beta-thalassemia major from Şanlıurfa province, Turkey.

OBJECTIVE The prevalence of β-thalassemia in Şanlıurfa province, Turkey is reported to be 2.6%-3.7%, whereas nation-wide the frequency of β-thalassemia is 2%. This study aimed to identify the most frequent β-thalassemia mutations in Şanlıurfa province. METHODS In total, 22 mutations were investigated in 115 pediatric patients with β-thalassemia using a commercially available reverse dot blot platform. RESULTS The study included 60 male and 55 female patients with a mean age of 7.3±4.6 years (range: 1-17 years). In total, 76% of the patients had consanguineous parents. In all, 16 different mutations were observed in the 115 patients. IVS-1-110 (G-A) (29.1%), IVS-1-1 (G-A) (13.9%), codon 39 (C>T) (10.4%), and codon 8 (-AA) (9.1%) accounted for 62.5% of all the β-thalassemia mutations, and 6% of the patients had 2 different thalassemia mutations. According to the present results, IVS-1-110 (G>A) was the most frequent mutation observed in the patients from Şanlıurfa province, as in other geographical regions of Turkey. In addition, the following 34 compound heterozygote mutant alleles were observed; IVS-1-1 (G>A)/IVS 2.848 (n=4), codon 39 (C>T)/codon 8 (-AA) (n=2), codon 6 (-A)/IVS 1.5 (G>C) (n=2), IVS-1-110 (G>A)/IVS-1-1 (G>A) (n=2), IVS-1-110 (G>A)/codon 8 (-AA) (n=1), IVS-1-110 (G>A)/codon 39 (C>T) (n=1), IVS-1-110 (G>A)/IVS-1-6 (T>C) (n=1), IVS-1-110 (G>A)/IVS-1-5 (G>C) (n=1), IVS-1-110 (G>A]/codon 8/9 (+G) (n=1), IVS-1-1 (G>A)/codon 39 (C>T) (n=1), and codon 8 (-AA)/IVS-1-5 (G>C) (n=1). The following β-globin gene promoter mutations were not observed; -101 (C>T), -87(C>T), -30 (T>A), codon 15 (TTG>TGA), codon 27 (G>T) Knossos, and IVS-1-116 (G>C). In all, 5 of the 115 patients (4.3%) had an unidentified mutation. CONCLUSION The present results illustrate the heterogeneity of β-thalassemia mutations in Şanlıurfa Province. The present findings may be of value for genetic counseling, and premarital and prenatal diagnosis in Şanlıurfa province.

miRNA-mediated apoptosis activation through TMEM 48 inhibition in A549 cell line

Lung has critic function in gas exchange, supplying oxygen to all cells. Rapid metastasis and the high rate of mortality characterises lung cancer. There are two types of this disease, small cell and non-small cell, which differs from each other according to histopathologic features. To date, many therapeutic approaches have been developed to destroy this deadly type of cancer, which one of them is mRNA targeted therapies through miRNA. miRNAs are 19-25 base paired molecules be able to suppress and destruct mRNA and found to be involved in development and progression of lung cancer. Transmembrane Protein 48 (TMEM48) is localised on nuclear pore complex and plays critic roles in nuclear traffic. Known that TMEM48 gene overexpressed in non-small lung cancer cells. Growing TMEM48 suppressed therapeutic studies indicated that decreased TMEM48 level might reveal a therapeutic effect for non-small cell lung cancers. TMEM48 studies based on the same strategy of gene-silencing, however, to our knowledge, any report has been published evaluates TMEM48s regulation by miRNAs. We aimed to clarify if miR-421 might be therapeutic player for non-small cancer cell lines (A549), hereby we suppressed TMEM48 by miR-421 and performed advanced molecular tests. Consequently, we recorded that while miR-421 is significantly suppressing TMEM48 expression; it increased apoptotic and tumor suppressor players CASPASE 3, PTEN and TP53 in A549 line, which is consistent with Annexin V - PI results: 30,6% of A549 observed to be apoptotic - 68,5% of A549 was in GO/G1. Our study indicated that miR-421 can suppress TMEM48 so that leads the cells to apoptosis. But it is not entirely clear how miR-421 triggers apoptosis and whether it interacts with the other cellular death pathways in A549.

Lack of Association between PTPN22 Gene +1858 C>T Polymorphism and Susceptibility to Generalized Vitiligo in a Turkish Population

Background Vitiligo is an autoimmune polygenic disorder characterized by loss of pigmentation due to melanocyte destruction. The PTPN22 gene +1858 C>T single nucleotide polymorphism (rs2476601) has been shown to be associated with various autoimmune disorders. Objective The aim of this study was to investigate whether the PTPN22 gene +1858 C>T single nucleotide polymorphism is associated with susceptibility to generalized vitiligo in a Turkish population. Methods One hundred and seven patients with generalized vitiligo, and one hundred and twelve gender-, age-, and ethnic-matched controls were enrolled in the study. Genotyping was done by polymerase chain reaction-restriction fragment length polymorphism. Results The PTPN22 +1858 C>T genotype and allele frequencies of the generalized vitiligo patients did not differ significantly from those of healthy controls. Conclusion We found no association between the PTPN22 +1858 C>T gene polymorphism and vitiligo susceptibility in Turkish generalized-vitiligo patients.