Molecular analyses of ADAMTS-1, -4, -5, and IL-17 a cytokine relationship in patients with ulcerative colitis.

BACKGROUND: Ulcerative colitis (UC) is a chronic inflammatory bowel disease that develops due to the impaired immune response in genetically susceptible individuals, and its etiopathogenesis is not fully elucidated. IL-17 A is a cytokine that is produced by a type of immune cell called Th17 cells and is involved in the immune response and inflammation. On the other hand, ADAMTS-1, -4, and - 5 are enzymes that are involved in the breakdown of extracellular matrix proteins, including proteoglycans, which are important components of the intestinal wall. This study aimed to evaluate the relationship between interleukin 17 (IL-17 A) cytokine, which plays a role in the pathogenesis of ulcerative colitis, and the inflammation-controlled a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-1, -4, and - 5 protein members. METHODS: Bowel tissue samples and blood serum from 51 patients with UC and 51 healthy controls were included in this study. mRNA expression levels of the ADAMTS-1, -4, -5, and IL-17 A were analyzed by RT-qPCR, and immunohistochemical analyses were performed to evaluate ADAMTS-1, -4, -5, and IL-17 A proteins in tissue samples. In addition, ELISA analysis determined serum levels of the ADAMTS-1, -4, -5, and IL-17 A. RESULTS: RT-qPCR results reveal that the expression of ADAMTS-1, -4, -5, and IL-17 A genes in the UC tissue samples were significantly high according to the control tissue samples. Also, ADAMTS-1, -4, -5, and IL-17 A proteins revealed enhanced expression pattern UC groups according to the control. Also, ADAMTS-1, -4, -5, and IL-17 A protein showed cytoplasmic localization patterns in both control and UC groups. The serum levels of ADAMTS-1,-5, and IL-17 A were significantly higher in UC samples than in the control group. CONCLUSIONS: We observed a positive correlation between the ADAMTS-1, -5 and IL17A cytokine expression in UC samples. These results provide a new understanding of controlling crucial ADAMTS family protein members by IL-17 A cytokines with UC.

Evaluation of the CRISPR/Cas9 directed mutant TP53 gene repairing effect in human prostate cancer cell line PC-3.

Prostate cancer is a common health problem among men worldwide and most of these prostate cancer cases are related to a dysfunctional mutant Tumor Protein p53 (TP53) gene. However, the CRISPR/Cas9 system can be used for repairing of a dysfunctional mutant TP53 gene in combination with donor single-stranded oligodeoxynucleotide (ssODN) via cells' own homology-directed repair (HDR) mechanism. In this study, we aimed to evaluate the CRISPR/Cas9 repairing efficiency on TP53 414delC (p.K139fs*31) null mutation, located in the TP53 gene, of human prostate cancer cell line PC-3 in combination with ssODNs. According to the next-generation sequencing results, TP53 414delC mutation was repaired with an efficiency of 19.95% and 26.0% at the TP53 414delC position with ssODN1 and ssODN2 accompanied by sgRNA2 guided CRISPR/Cas9, respectively. Besides, qPCR and immunofluorescence analysis showed that PC-3 cells, the TP53 414delC mutation of which were repaired, expressed wild type p53 again. Also, significantly increased number of apoptotic cells, driven by the repaired TP53 gene were detected compared to the control cells by flow cytometry analysis. As a result, sgRNA2 guided CRISPR/Cas9 system accompanied by ssODN was shown to effectively repair the TP53 414delC gene region and inhibit the cell proliferation of PC-3 cells. Therefore, the effects of the TP53 414delC mutation repairment in PC-3 cells will be investigated in the in vivo models for tumor clearance analysis in the near future.

Clinicopathological characteristics and mutation profile of BRAF and NRAS mutation in cutaneous melanomas in the Western Turkish population

Background/aim: Malignant melanoma is the most common cause of death due to skin cancers. The most common mutations in RAFRAS pathway from tumor oncogenes are BRAF and NRAS. In this study, we analyzed the frequency of BRAF and NRAS gene mutations and investigated their association with clinicopathological features of melanomas in the Turkish population. Materials and methods: 65 primary cutaneous melanoma were included in the study. The mutations were evaluated with real-time PCRbased PCR-array through allele-specific amplification, and the results were correlated with various clinicopathological characteristics. Results: 52.3% of the patients were female and 47.7% were male. The mean age of the patients with a mutation was lower than those without mutation. 16 patients had BRAF mutation. 12 patients had NRAS mutation. NRAS mutation was statistically more common in men (P = 0.036). The number of mitoses increased with the increase of the tumor thickness (P = 0.003). There was more mitosis in the presence of ulceration (P = 0.05). A total of 41.7% of NRAS mutations had adjuvant chemotherapy. Conclusion: We found lower mutation rate when compared to regional studies. NRAS mutation was common in men. This is the first study from our region evaluating the prognostic value of clinical stage and necessity of adjuvant treatment with the presence of BRAF and NRAS mutations.

Relationship Between Chronic Tinnitus and Glial Cell Line-Derived Neurotrophic Factor Gene rs3812047, rs1110149, and rs884344 Polymorphisms in a Turkish Population.

Glial cell line-derived neurotrophic factor (GDNF) plays a key role in early development of central auditory pathway and the inner ear. However, the auditory pathway studies of GDNF gene polymorphisms are scarce in the literature, and the studies especially associated with tinnitus are limited. Our study aimed to identify whether GDNF gene polymorphisms play any roles in the pathophysiology of tinnitus by investigating the relationship between tinnitus and GDNF polymorphisms. A total of 52 patients with chronic tinnitus and ages ranging from 18 to 55 were admitted to the Ear-Nose-Throat outpatient clinic of Celal Bayar University Medical Faculty Hospital of Manisa, Turkey and constituted the study group. Another 42 patients of the same age range, without tinnitus symptoms and lacking any systemic disease, were also admitted to the clinic and formed the control group. The tympanometric, audiological, and psychoacoustic assessments of the subjects were performed. Deoxyribonucleic acid samples obtained using venous blood taken for routine inspections were used to investigate GDNF gene polymorphisms (rs884344, rs3812047, and rs1110149) by polymerase chain reaction-based restriction fragment length polymorphism method. No correlation could be detected between GDNF rs884344 and rs3812047 polymorphisms and subjects with tinnitus (p > 0.05). Heterozygosity was significantly lower for GDNF rs1110149 polymorphism in tinnitus subjects compared to the controls (p < 0.05). However, the allele frequencies for all 3 polymorphisms were not significantly different between tinnitus and control groups (p > 0.05). Failure to detect correlations between tinnitus and GDNF gene polymorphisms suggests this may be due to the fact that the GDNF gene has a variable expression pattern in different tissues and pathologies. Therefore, the study should be improved and its scope should be expanded by including a larger group of patients and different tissues to investigate the expression pattern of GDNF.

Genes predisposing tunneled catheter thrombosis in hemodialysis patients.

Background: This study aims to investigate the association of genes predisposing thrombophilia with tunneled catheter thrombosis in hemodialysis patients. Methods: Between October 2018 and December 2020, we compared the frequencies of genetic polymorphisms causing thrombophilia, including prothrombin G20210A, factor V Leiden, methylene tetrahydrofolate reductase (MTHFR) C677T, MTHFR A1298C, plasminogen activator inhibitor (PAI), factor XIII V34L and clinical characteristics of 52 patients with a history of 2 tunneled2 tunneled catheter thrombosis occlusions within a year (Group 1; 24 males, 28 females; mean age: 62±8.9 years; range, 45 to 77 years), 52 patients who underwent their first tunneled catheter thrombosis insertion (Group 2; 29 males, 23 females; mean age: 63±15.2 years; range, 22 to 87 years), and 51 healthy controls (Group 3; 26 males, 25 females; mean age: 34±9.2 years; range, 19 to 54 years). Results: Groups 1 and 2 carried the MTHFR A1298C (p=0.048) and compound heterozygous MTHFR A1298C and C677T (p=0.048) polymorphisms more frequently than Group 3. However, subgroup analysis results were not statistically significant. The other polymorphisms were distributed similarly in all three groups. The MTHFR polymorphisms had a weak effect on tunneled hemodialysis catheter thrombosis in neural network analysis. Conclusion: Our study results indicated that there was a concomitance of MTHFR polymorphisms with hemodialysis-dependent chronic kidney disease. The MTHFR A1298C and compound heterozygous MTHFR polymorphisms may be associated with tunneled hemodialysis catheter thrombosis. Thrombophilia gene screening may be recommended in hemodialysis patients undergoing tunneled hemodialysis catheter thrombosis at least twice in a year.

A new clinical entity in T704M mutation in periodic paralysis.

Periodic paralyses (PPs) are a group of rare disorders characterized by episodic, sudden-onset, flaccid paralysis of skeletal muscles usually resulting in complete recovery after the attacks. PPs are caused by abnormal, mostly potassium-sensitive excitability of the muscle tissue. Hypokalemic and hyperkalemic periodic paralysis (HypoKPP and HyperKPP) have been described according to their characteristic phenotypes and the serum potassium level during the attacks of weakness. The T704M mutation on the SCN4A gene is the most common mutation in HyperKPP. Different mutations of the SCN4A gene have also been reported in some cases of HypoKPP. In this study, a large Turkish family carrying the T704M mutation on the SCN4A gene with HypoKPP disease was examined. A similar history was noted in a total of 17 subjects in the pedigree. SCN4A gene of the patients was sequenced with Sanger sequencing. In this study, this mutation was associated with a HypoKKP diagnosis for the first time in the literature. The symptoms of hallucination and diplopia seen in patients had also never been indicated in the literature before. This report expands the phenotypic variability of the T704M mutation, further confirming the lack of genotype-phenotype correlation in SCN4A mutations.

Coinheritance of novel mutations in NAGLU causing mucopolysaccharidosis type IIIB and in DDHD2 causing spastic paraplegia54 in a Turkish family.

Mucopolysaccharidosis type IIIB (MPSIIIB) is one of the lysosomal storage diseases, clinically related to developmental delay in the early phase and loss of skills in the late phases of the disease. The disease is caused by homozygous mutations in the NAGLU gene. Spastic paraplegia54 (SPG54) is a neurodegenerative disorder caused by homozygous mutations in the DDHD2 gene. Clinical features are progressive spasticity and weakness in the lower limbs and corpus callosum agenesis. We report on two siblings in a consanguineous family, presenting both the clinical and molecular diagnoses of MPSIIIB and SPG54 with novel mutations by using whole exome sequencing (WES). This interesting finding shows that we should be aware of the importance of using WES for diagnosing rare diseases in consanguineous families.

No association of interleukin-6 gene polymorphism (-174 G/C) with premature coronary artery disease in a Turkish cohort.

OBJECTIVES: Interleukin-6 (IL-6) may contribute to the inflammatory response by activating endothelial cells and stimulating the synthesis of fibrinogen. It might thus be important in the pathogenesis of inflammation associated with coronary artery disease (CAD). Several studies suggested that the -174 C allele was associated with an increased prevalence of coronary heart disease. The aim of this study was to investigate further the association of the IL-6 -174 G/C allele status with premature CAD. METHODS: A total of 120 patients and 105 controls were included in the study. The IL-6 -174 G/C polymorphism was genotyped using PCR-restriction fragment length polymorphism. RESULTS: The genotype distribution of the -174 G/C polymorphism was not different in premature CAD patients (GG: 53%; GC: 42.6%; CC: 4.3%) and controls (GG: 54.3%; GC: 39%; CC: 6.7%) (P=0.72). The prevalence of the C allele was 25.6% in patients and 26.1% in controls. By multiple regression analysis, family history, smoking, diabetes, and hypertension were independent risk factors of premature CAD, but not IL-6 genotype. CONCLUSIONS: We conclude that the IL-6 -174 G/C polymorphism is not associated with the risk of premature CAD, and does not contribute to cardiovascular risk stratification.

A novel association between TGFb1 and ADAMTS4 in coronary artery disease: A new potential mechanism in the progression of atherosclerosis and diabetes.

OBJECTIVE: Coronary artery disease is characterized by atherosclerosis in the vessel wall. Recently, it has been thought that increasing LDL-binding capacity of subendothelial proteoglycan fragments that are formed by protease activity can be responsible for the initiation of atherosclerosis. ADAMTS4 is a member of the versican-degrading proteinases. In vitro studies demonstrated that TGFb inhibits the expression of ADAMTS4 in macrophages. In this study, we aimed to investigate the role and association between TGFb1 and ADAMTS4 in coronary artery disease. METHODS: A total of 84 cases with atheroma plaque and 72 controls without plaque were analyzed. The severity of disease was determined by Gensini score. TGFb1 gene polymorphisms were genotyped by the PCR-RFLP method. TGFb1 and ADAMTS4 serum levels were measured by ELISA method. Statistical analyses of genotypes and their relationship with serum levels were performed by chi-square, student t test and ANOVA. RESULTS: ADAMTS4 levels were higher in cases compared with controls (p<0.05). In the patient group, ADAMTS4 levels were higher than in controls and correlated with TGFb1 serum levels (r=0.29; p<0.05) and severity of disease (r=0.20; p<0.05). The TGFb1 gene CCA haplotype was associated with 3.3-fold increase in coronary artery disease (OR=3.26 95% CI 1.22-8.68; p<0.05). Unexpectedly, ADAMTS4 serum levels were also higher in diabetic cases (p=0.05). CONCLUSION: This study has demonstrated that ADAMTS4 may be responsible for the pathogenesis of atherosclerosis. This is the first report about the association between ADAMTS4 and TGFb1 serum levels in the progression of atherosclerosis in CAD. Furthermore, it is seen that TGFb1 haplotype can cause a genetic susceptibility to CAD in the Turkish population. To our knowledge, this is also the first report suggesting higher serum ADAMTS4 levels in diabetic patients.

Relationship between ace genotype and short duration aerobic performance development.

We have previously demonstrated that, ACE D allele may be related with a better performance in short duration aerobic endurance in a homogeneous cohort with similar training backgrounds. We aimed to study the variation in the short-duration aerobic performance development amongst ACE genotypes in response to identical training programs in homogeneous populations. The study group consisted of 186 male Caucasian non-elite Turkish army recruits. All subjects had undergone an identical training program with double training session per day and 6 days a week for 6 months. Performances for middle distance runs (2,400 m) were evaluated on an athletics track before and after the training period. ACE gene polymorphisms were studied by PCR analysis. The distribution of genotypes in the whole group was 16.7% II, n=31; 46.2% ID, n=86; 37.1% DD, n=69. Subjects with ACE DD genotype had significantly higher enhancement than the ID (P<0.01) and II (P<0.05) genotype groups. Around 2,400 m performance enhancement ratios showed a linear trend as ACE DD>ACE ID>ACE II (P value for Pearson chi2=0.461 and P value for linear by linear association=0.001). ACE DD genotype seems to have an advantage in development in short-duration aerobic performance. This data in unison with the data that we have obtained from homogenous cohorts previously is considered as an existence of threshold for initiation of ACE I allele effectiveness in endurance performance. This threshold may be anywhere between 10 and 30 min with lasting maximal exercises.