Aris Cakiris

Genome-wide association study identifies variants in the MHC class I, IL10, and IL23R-IL12RB2 regions associated with Behcet's disease

Behçets disease is a genetically complex disease of unknown etiology characterized by recurrent inflammatory attacks affecting the orogenital mucosa, eyes and skin. We performed a genome-wide association study with 311,459 SNPs in 1,215 individuals with Behçets disease (cases) and 1,278 healthy controls from Turkey. We confirmed the known association of Behçets disease with HLA-B*51 and identified a second, independent association within the MHC Class I region. We also identified an association at IL10 (rs1518111, P = 1.88 × 10−8). Using a meta-analysis with an additional five cohorts from Turkey, the Middle East, Europe and Asia, comprising a total of 2,430 cases and 2,660 controls, we identified associations at IL10 (rs1518111, P = 3.54 × 10−18, odds ratio = 1.45, 95% CI 1.34–1.58) and the IL23R-IL12RB2 locus (rs924080, P = 6.69 × 10−9, OR = 1.28, 95% CI 1.18–1.39). The disease-associated IL10 variant (the rs1518111 A allele) was associated with diminished mRNA expression and low protein production.

Association of Familial Mediterranean Fever-Related MEFV Variations With Ankylosing Spondylitis

OBJECTIVE The pathogenesis of ankylosing spondylitis (AS) has a strong genetic contribution. Familial Mediterranean fever (FMF) is an autosomal recessively inherited autoinflammatory disorder caused by MEFV gene missense variations, and a clinical association between FMF and AS has been reported previously. The aim of this study was to analyze the association of common MEFV variations (M694V, M680I, V726A, and E148Q) with AS in a group of Turkish patients. METHODS The study group comprised 193 patients with AS and 103 matched healthy control subjects. All individuals were genotyped for 4 MEFV variations and HLA-B27 using genomic DNA, and association of the variations with the clinical and laboratory features of the patients was analyzed. RESULTS The MEFV missense variations were significantly more frequent in patients with AS (22.3%) compared with healthy control subjects (9.7%; odds ratio [OR] 2.67, 95% confidence interval [95% CI] 1.28-5.56). This difference was more prominent for exon 10 variations (M694V, V726A, M680I) (OR 3.75, 95% CI 1.41-9.97), especially for the most-penetrant variation M694V (OR 4.73, 95% CI 1.39-16.12). MEFV variations were more frequent in HLA-B27-negative patients with AS, and the difference was statistically significant in patients carrying exon 10 variants. CONCLUSION FMF-related MEFV variations are associated with AS, and these variations may contribute to the pathogenesis of AS, especially in populations in which the prevalence of FMF is high.

Characterization of H3K9me3- and H4K20me3-associated circulating nucleosomal DNA by high-throughput sequencing in colorectal cancer

Modified histone tails in nucleosomes circulating in the blood bear the potential as cancer biomarkers. Recently, using chromatin immunopecipitation (ChIP)-related quantitative PCR, we described reduced plasma levels of the two pericentric heterochromatin-specific histone methylation marks H3K9me3 and H4K20me3 in patients with colorectal cancer (CRC). Here, by utilizing ChIP-related high-throughput sequencing, we further characterized these modifications in circulation. Plasma DNA from nucleosomes immunoprecipitated by H3K9me3- and H4K20me3-specific antibodies from patients with CRC (N = 15) and healthy subjects (N = 15) was subjected to the Roche 454 FLX sequencing, and the generated array of ChIP-enriched sequences were compared to the human reference genome. The total number of nucleosomes, of sequence reads and of diverse DNA repetitive elements were statistically compared between the study groups. Total nucleosome amount was not different in both groups. Concerning both histone modifications, lower numbers of sequence reads were detected in CRC patients as compared with healthy controls (medians in H3K9me3: 32 vs. 61; p < 0.01; in H4K20me3: 54 vs. 88; p < 0.01). Size of fragments was not different in both groups. Most abundant sequences were repetitive LINE and SINE elements while simple repeats, LTR, DNA, SAT, and low complexity elements were less frequent. Best discrimination between both groups was achieved by total number of H3K9me3 reads (AUC 0.90) and H3K9me3 LINE elements L1 (AUC 0.93) und L2 (AUC 0.91). The present results confirm earlier findings of lower H3K9me3 levels in CRC and show LINE elements to be the most frequent and best discriminative markers on modified histones.

Whole mitochondrial genome analysis of a family with NARP/MILS caused by m.8993T>C mutation in the MT-ATP6 gene.

Mutations in mitochondrial DNA (mtDNA) encoded nucleotide 8993 can cause NARP syndrome (neuropathy, ataxia, and retinitis pigmentosa) or MILS (maternally inherited Leigh syndrome). The rare T8993C mutation in the MT-ATP6 gene is generally considered to be clinically milder, but there is marked clinical heterogeneity ranging from asymptomatic carriers to fatal infantile Leigh syndrome. Clinical heterogeneity has mostly been attributed to mtDNA heteroplasmy, but environmental, autosomal, tissue-specific factors, nuclear modifier genes, and mtDNA variations may also modulate disease expression. Here, we report the results of whole mitochondrial genome analysis of a family with m.8993T>C mutation in the MT-ATP6 gene and associated with NARP/MILS, and discuss the familial inheritance, effects of variation in combinations and heteroplasmy levels on the clinical findings. The whole mitochondrial genome was sequenced with ~182× average depth of coverage per sample with next-generation sequencing technology. Thus, all heteroplasmic (>%10) and homoplasmic variations were determined (except for 727C insertion) and classified according to the associations with mitochondrial diseases.

Investigation of Arg399Gln and Arg194Trp polymorphisms of the XRCC1 (x-ray cross-complementing group 1) gene and its correlation to sister chromatid exchange frequency in patients with chronic lymphocytic leukemia.

Polymorphisms of the x-ray repair cross-complementing group 1 (XRCC1) gene have been reported to be associated with various forms of cancer. We evaluated the possible effects of the Arg194Trp and the Arg399Gln polymorphisms on the risk for chronic lymphocytic leukemia (CLL) in 73 patients and 50 controls. We also analyzed their relation to frequency of sister chromatid exchange (SCE). With respect to codon 194, the allelic frequency of the Arg194Trp polymorphism did not significantly differ between the 2 groups. The proportion of individuals carrying the Arg194Trp polymorphism was not different in the 2 groups. With respect to codon 399, the proportion of the individuals carrying the Arg399Gln allele (90% vs 62%; p=0.000; odds ratio [OR], 5.779; 95% confidence interval [CI], 2.2-15.183) and the allelic frequency of the Arg399Gln polymorphism (56% vs 36%; p=0.002; OR, 2.278; 95% CI, 1.350-3.843) was significantly higher in the patient group. The frequency of the Arg/Gln genotype was significantly higher in the patient group (68.50% vs 52%; p=0.049; OR, 2.007; 95% CI, 0.955-4.217). The mean SCE frequency in the patient group was significantly higher (9.2±4 vs 7.5±2; p=0.02). When different compound genotypes were compared, the coexistence of Arg/Arg genotype in codon 194 with Arg/Arg genotype in codon 399 was significantly more frequent in the control group (30% vs 9%; p=0.004; OR, 0.247; 95% CI, 0.092-0.664). Within the patient group, SCE frequency did not differ between patients with various genotypes. The Arg399Gln polymorphism may be etiologically associated with CLL; however, it does not seem to increase SCE frequency.

VEGF-A and FGF gene therapy accelerate healing of ischemic colonic anastomoses (experimental study)

BACKGROUND Reducing ischemic damage is one of the goals of surgery. The aim of this study was to apply human VEGF-A and FGF-2 DNA-mediated gene therapy in order to identify their effects in the healing of ischemic colon anastomoses and eliminating the negative effects of ischemia. METHODS Forty male Wistar albino rats weighing 250-280 g were divided into five equal groups (n = 8) as follows: group 1: control, ischemic left colonic anastomosis; group; 2: ischemic left colonic anastomosis with control plasmid delivery; group 3: ischemic left colonic anastomosis with VEGF plasmid delivery; group 4: ischemic left colonic anastomosis with FGF plasmid delivery; group 5: ischemic left colonic anastomosis with VEGF and FGF plasmid delivery. All rats were sacrificed on the 4th postoperative day. Anastomosis burst pressures were measured for mechanical examination of anastomosis. Tissue hydroxyprolin, VEGF and FGF levels were determined as biochemical parameters. Necrosis, epithelisation, inflammatory processes, fibroblastic activity, collagen deposition and neovascularisation at the anastomic site were studied. RESULTS VEGF, FGF and combined therapy significantly accelerated many of the histological parameters of healing, including fibroblast activation, collagen deposition, and angiogenesis, and augmented the levels of hydroxyproline and bursting pressure. CONCLUSIONS This is the first study to use gene therapy with growth factors for the healing of ischemic colonic anastomosis. This therapy can be effectively used in increasing ischemic anastomosis wound healing.

Effects of curcumin on proinflammatory cytokines and tissue injury in the early and late phases of experimental acute pancreatitis

BACKGROUND & AIMS Acute pancreatitis (AP) varies from mild to severe necrotizing changes with high mortality. The objective of the current study was to investigate the effects of curcumin on tissue injury and proinflammatory cytokines in the early and late phases of AP. METHODS AP was induced by sodium taurocholate in rats (n = 140). First group was left untreated. Group II received 100 mg/kg curcumin daily starting 20 days before AP induction. The rats were allocated into 7 sub-groups (n:5) and were sacrificed at 2, 6, 12, 24, 72, 144 and 288 h following the induction of AP. Blood and pancreatic tissue samples were collected for biochemical and histopathologic evaluations and the assessment of protein and mRNA levels, as well. RESULTS Curcumin decreased total histopathologic scores in comparison with those of the taurocholate group (P < 0.05). Curcumin increased Caspase-3 activity and decreased trypsin activity, while inhibited nuclear factor-κ (NF-κB) at all time points (P < 0.05) and moreover reduced activator protein-1 (AP-1). Curcumin decreased chemokine (except for 288 h), TNF-α (except for 2 and 24 h), IL-6 (except for 2, 6 and 288 h) and iNOS (except for 144 and 288 h) mRNA levels (P < 0.05). Curcumin serum nitric oxide (NO) (except for 144 and 288 h) levels were reduced, as well. CONCLUSIONS In conclusion, curcumin reduced tissue injury, trypsin activation and inhibited NF-κB and AP-1. However TNF-α, IL-6 and iNOS and NO were not inhibited at all time points. Therefore no direct correlation was detected in the subgroups between tissue injury, proinflammatory cytokines and oxidative enzymes.

Draft Genome Sequence of Halomonas smyrnensis AAD6T

Halomonas smyrnensis AAD6(T) is a Gram-negative, aerobic, exopolysaccharide-producing, and moderately halophilic bacterium that produces levan, a fructose homopolymer with many potential uses in various industries. We report the draft genome sequence of H. smyrnensis AAD6(T), which will accelerate research on the rational design and optimization of microbial levan production.

A genome-wide analysis of lentivector integration sites using targeted sequence capture and next generation sequencing technology

One application of next-generation sequencing (NGS) is the targeted resequencing of interested genes which has not been used in viral integration site analysis of gene therapy applications. Here, we combined targeted sequence capture array and next generation sequencing to address the whole genome profiling of viral integration sites. Human 293T and K562 cells were transduced with a HIV-1 derived vector. A custom made DNA probe sets targeted pLVTHM vector used to capture lentiviral vector/human genome junctions. The captured DNA was sequenced using GS FLX platform. Seven thousand four hundred and eighty four human genome sequences flanking the long terminal repeats (LTR) of pLVTHM fragment sequences matched with an identity of at least 98% and minimum 50 bp criteria in both cells. In total, 203 unique integration sites were identified. The integrations in both cell lines were totally distant from the CpG islands and from the transcription start sites and preferentially located in introns. A comparison between the two cell lines showed that the lentiviral-transduced DNA does not have the same preferred regions in the two different cell lines.

No association of the TLR2 gene Arg753Gln polymorphism with rheumatic heart disease and Behçet’s disease

Behçet’s disease (BD) is a multisystem inflammatory disorder of unknown etiology, and infections with different microorganisms including streptococci have been claimed as triggers of inflammatory attacks in BD pathogenesis. Toll-like receptor 2 (TLR2) has been known to recognize several microbial antigens including that of streptococci, and TLR2 gene Arg753Gln polymorphism has been reported to be strongly associated with acute rheumatic fever with an odds ratio of 100. This study aimed to investigate the TLR2 gene Arg753Gln polymorphism in a group of patients with BD and rheumatic heart disease (RHD) and to analyze the role of genotyping errors resulting from duplicated gene segments. The study group consisted of 211 patients with BD, 95 patients with RHD, and 94 matched Turkish healthy controls. Because of the duplicated exon 3 in 23-kb upstream of the TLR2 gene, genotyping for the Arg753Gln polymorphism with polymerase chain reaction–restriction fragment length polymorphism method was carried out using a new set of primers and PstI restriction enzyme. TLR2 gene Gln753 allele was observed in two of 211 (1.0%) patients with BD, five of 95 (5.3%) patients with RHD, and two of 94 (2.1%) healthy controls. All patients and controls were found to be heterozygous for Arg753Gln polymorphism, except one patient with BD, who was homozygous for Gln753. Although a slight increase of heterozygosity was noted in patients with RHD, no statistically significant difference was observed in the distribution of Arg753Gln polymorphism in BD and RHD compared to healthy controls. In conclusion, TLR2 gene Arg753Gln polymorphism is not associated with BD nor with RHD; and a duplicated region of the TLR2 exon 3 located 23-kb upstream of the polymorphic region may explain contradictory association findings described so far.