Jurgita Skieceviciene

Association Between Variants of PRDM1 and NDP52 and Crohn's Disease, Based on Exome Sequencing and Functional Studies

BACKGROUND & AIMS Genome-wide association studies (GWAS) have identified 140 Crohns disease (CD) susceptibility loci. For most loci, the variants that cause disease are not known and the genes affected by these variants have not been identified. We aimed to identify variants that cause CD through detailed sequencing, genetic association, expression, and functional studies. METHODS We sequenced whole exomes of 42 unrelated subjects with CD and 5 healthy subjects (controls) and then filtered single nucleotide variants by incorporating association results from meta-analyses of CD GWAS and in silico mutation effect prediction algorithms. We then genotyped 9348 subjects with CD, 2868 subjects with ulcerative colitis, and 14,567 control subjects and associated variants analyzed in functional studies using materials from subjects and controls and in vitro model systems. RESULTS We identified rare missense mutations in PR domain-containing 1 (PRDM1) and associated these with CD. These mutations increased proliferation of T cells and secretion of cytokines on activation and increased expression of the adhesion molecule L-selectin. A common CD risk allele, identified in GWAS, correlated with reduced expression of PRDM1 in ileal biopsy specimens and peripheral blood mononuclear cells (combined P = 1.6 × 10(-8)). We identified an association between CD and a common missense variant, Val248Ala, in nuclear domain 10 protein 52 (NDP52) (P = 4.83 × 10(-9)). We found that this variant impairs the regulatory functions of NDP52 to inhibit nuclear factor κB activation of genes that regulate inflammation and affect the stability of proteins in Toll-like receptor pathways. CONCLUSIONS We have extended the results of GWAS and provide evidence that variants in PRDM1 and NDP52 determine susceptibility to CD. PRDM1 maps adjacent to a CD interval identified in GWAS and encodes a transcription factor expressed by T and B cells. NDP52 is an adaptor protein that functions in selective autophagy of intracellular bacteria and signaling molecules, supporting the role of autophagy in the pathogenesis of CD.

Geographical patterns of the standing and active human gut microbiome in health and IBD

Objective A global increase of IBD has been reported, especially in countries that previously had low incidence rates. Also, the knowledge of the human gut microbiome is steadily increasing, however, limited information regarding its variation on a global scale is available. In the light of the microbial involvement in IBDs, we aimed to (1) identify shared and distinct IBD-associated mucosal microbiota patterns from different geographical regions including Europe (Germany, Lithuania) and South Asia (India) and (2) determine whether profiling based on 16S rRNA transcripts provides additional resolution, both of which may hold important clinical relevance. Design In this study, we analyse a set of 89 mucosal biopsies sampled from individuals of German, Lithuanian and Indian origins, using bacterial community profiling of a roughly equal number of healthy controls, patients with Crohns disease and UC from each location, and analyse 16S rDNA and rRNA as proxies for standing and active microbial community structure, respectively. Results We find pronounced population-specific as well as general disease patterns in the major phyla and patterns of diversity, which differ between the standing and active communities. The geographical origin of samples dominates the patterns of β diversity with locally restricted disease clusters and more pronounced effects in the active microbial communities. However, two genera belonging to the Clostridium leptum subgroup, Faecalibacteria and Papillibacter, display consistent patterns with respect to disease status and may thus serve as reliable ‘microbiomarkers’. Conclusions These analyses reveal important interactions of patients’ geographical origin and disease in the interpretation of disease-associated changes in microbial communities and highlight the added value of analysing communities on both the 16S rRNA gene (DNA) and transcript (RNA) level.

Gene Polymorphisms of Micrornas in Helicobacter pylori-Induced High Risk Atrophic Gastritis and Gastric Cancer

Background and aims MicroRNAs (miRNAs) are known for their function as translational regulators of tumor suppressor or oncogenes. Single nucleotide polymorphisms (SNPs) in miRNAs related genes have been shown to affect the regulatory capacity of miRNAs and were linked with gastric cancer (GC) and premalignant gastric conditions. The purpose of this study was to evaluate potential associations between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of GC or high risk atrophic gastritis (HRAG) in European population. Methods Gene polymorphisms were analyzed in 995 subjects (controls: n = 351; GC: n = 363; HRAG: n = 281) of European descent. MiR-27a T>C (rs895819), miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913), miR-492 G>C (rs2289030) and miR-608 C>G (rs4919510) SNPs were genotyped by RT-PCR. Results Overall, SNPs of miRNAs were not associated with the presence of GC or HRAG. We observed a tendency for miR-196a-2 CT genotype to be associated with higher risk of GC when compared to CC genotype, however, the difference did not reach the adjusted P-value (odds ratio (OR) - 1.46, 95% confidence interval (CI) 1.03-2.07, P = 0.032). MiR-608 GG genotype was more frequent in GC when compared to controls (OR −2.34, 95% CI 1.08–5.04), but significance remained marginal (P = 0.029). A similar tendency was observed in a recessive model for miR-608, where CC + CG vs GG genotype comparison showed a tendency for increased risk of GC with OR of 2.44 (95% CI 1.14–5.22, P = 0.021). The genotypes and alleles of miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 SNPs had similar distribution between histological subtypes of GC and were not linked with the presence of diffuse or intestinal-type GC. Conclusions Gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of HRAG, GC or different histological subtypes of GC in European subjects.

Epigenetic silencing of miR-137 is a frequent event in gastric carcinogenesis.

MicroRNAs (miRNA) are involved in posttranscriptional regulation of gene expression and are dysregulated during carcinogenesis. CpG island methylation of miR‐137 is a common event in different cancers; however, the role of miR‐137 in gastric cancer (GC) remains largely unexplored. In this study we aimed to characterize the epigenetic alterations of miR‐137 in gastric carcinogenesis. We analyzed total 295 tissues including paired primary gastric cancer (T‐GC) with corresponding adjacent gastric mucosa (N‐GC), paired primary colorectal cancer (CRC) tissues with corresponding non‐tumorous mucosa, gastric tissues from controls (N), and patients with chronic/atrophic gastritis (CG) with and without Helicobacter pylori infection. Bisulfite pyrosequencing and TaqMan RT‐PCR were used to analyze miR‐137 methylation and expression, respectively. Survival differences were evaluated using Kaplan‐Meier analyses. miR‐137 CpG island methylation was more frequent in tumorous compared to non‐tumorous conditions and higher in CRC than in GC. In comparison to N‐GC, miR 137 methylation level was lower in N and CG tissues, which correlates with Correas cascade. MiR‐137 methylation inversely correlates with global LINE‐1 methylation and miR‐137 expression. miR‐137 methylation was higher in intestinal type GC compared to diffuse one, and higher in antrum compared to cardia and corpus, however, miR‐137 methylation was associated with worse prognosis in diffuse, but not in intestinal type of GC. The expression in colon was significantly higher compared to any gastric tissues suggesting functional difference. In summary, miR‐137 methylation is a frequent event in gastrointestinal cancers which occurs early in stepwise manner during gastric carcinogenesis and inversely correlates with global methylation.

Lack of association between miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608 gene polymorphisms and colorectal cancer.

Colorectal cancer (CRC) is one of the most common cancers worldwide with high mortality rates. MicroRNAs (miRNAs) have an established role in the development of different cancers. Single nucleotide polymorphisms (SNPs) in miRNA related genes were linked with various gastrointestinal malignancies. However, the data on association between miRNA SNPs and CRC development are inconsistent. The aim of the present study was to evaluate the association between miRNA-related gene polymorphisms (miR-27a, miR-146a, miR-196a-2, miR-492 and miR-608) and the presence of CRC in European population. Gene polymorphisms were analyzed in 621 subjects (controls: n = 428; CRC: n = 193). MiR-27a T>C (rs895819), miR-146a G>C (rs2910164), miR-196a-2 C>T (rs11614913), miR-492 G>C (rs2289030) and miR-608 C>G (rs4919510) SNPs were genotyped by RT-PCR. Overall, all genotypes and alleles of miRNA SNPs were distributed equally between control and CRC groups. We observed a tendency for miR-146a C allele to be associated with lower risk of CRC when compared to G allele, however, the difference did not reach the adjusted P-value (odds ratio (OR) = 0.68, 95% confidence interval (CI) 0.49–0.95, P = 0.025). In conclusion, gene polymorphisms of miR-27a, miR-146a, miR-196a-2, miR-492, miR-492a and miR-608 were not associated with the presence of CRC in European subjects.

Analysis of Deregulated microRNAs and Their Target Genes in Gastric Cancer.

Background MicroRNAs (miRNAs) are widely studied non-coding RNAs that modulate gene expression. MiRNAs are deregulated in different tumors including gastric cancer (GC) and have potential diagnostic and prognostic implications. The aim of our study was to determine miRNA profile in GC tissues, followed by evaluation of deregulated miRNAs in plasma of GC patients. Using available databases and bioinformatics methods we also aimed to evaluate potential target genes of confirmed differentially expressed miRNA and validate these findings in GC tissues. Methods The study included 51 GC patients and 51 controls. Initially, we screened miRNA expression profile in 13 tissue samples of GC and 12 normal gastric tissues with TaqMan low density array (TLDA). In the second stage, differentially expressed miRNAs were validated in a replication cohort using qRT-PCR in tissue and plasma samples. Subsequently, we analyzed potential target genes of deregulated miRNAs using bioinformatics approach, determined their expression in GC tissues and performed correlation analysis with targeting miRNAs. Results Profiling with TLDA revealed 15 deregulated miRNAs in GC tissues compared to normal gastric mucosa. Replication analysis confirmed that miR-148a-3p, miR-204-5p, miR-223-3p and miR-375 were consistently deregulated in GC tissues. Analysis of GC patients’ plasma samples showed significant down-regulation of miR-148a-3p, miR-375 and up-regulation of miR-223-3p compared to healthy subjects. Further, using bioinformatic tools we identified targets of replicated miRNAs and performed disease-associated gene enrichment analysis. Ultimately, we evaluated potential target gene BCL2 and DNMT3B expression by qRT-PCR in GC tissue, which correlated with targeting miRNA expression. Conclusions Our study revealed miRNA profile in GC tissues and showed that miR-148a-3p, miR-223-3p and miR-375 are deregulated in GC plasma samples, but these circulating miRNAs showed relatively weak diagnostic performance as sole biomarkers. Target gene analysis demonstrated that BCL2 and DNMT3B expression in GC tissue correlated with their targeting miRNA expression.

Influence of NADPH oxidase on inflammatory response in primary intestinal epithelial cells in patients with ulcerative colitis

BackgroundThe aim of this study is to evaluate the role of NADPH oxidase in primary intestinal epithelial cells during the active phase of UC.MethodsThe primary human colonic epithelial cells were isolated from 19 patients with mild to moderate inflammatory activity of UC and 14 controls using chelation method. The cells were cultivated under the effect of mediators. Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorimetrically using Amplex Red. Production of TNF-α cytokine by the colonic epithelial cells was analysed by ELISA.ResultsThe results of our study showed that unstimulated cells of UC patients had a decreased viability, increased ROS production, but similar TNF-α level when compared to the controls. Stimulation with LPS increased hydrogen peroxide and TNF-α level in the UC group. Treatment of colonic epithelial cells with NADPH oxidase inhibitor increased cell viability decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from UC patients.ConclusionsOur study showed that bacterial endotoxins induced NADPH oxidase activation in the colonic epithelial cells. Moreover, we revealed that treatment with NADPH oxidase inhibitors had a protective effect against pro-inflammatory action of LPS in human colonic epithelium cells during inflammation.

Protective action of NADPH oxidase inhibitors and role of NADPH oxidase in pathogenesis of colon inflammation in mice

AIM To investigate the role of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in colon epithelial cells in the pathogenesis of acute and chronic colon inflammation in a mouse model of dextran sulphate sodium (DSS)-induced colitis. METHODS Balb/c mice were divided into three groups: 8 mice with acute DSS-induced colitis (3.5% DSS solution; 7 d), 8 mice with chronic DSS-induced colitis (3.5% DSS solution for 5 d + water for 6 d; 4 cycles; total: 44 d) and 12 mice without DSS supplementation as a control group. Primary colonic epithelial cells were isolated using chelation method. The cells were cultivated in the presence of mediators (lipopolysaccharide (LPS), apocynin or diphenyleneiodonium). Viability of cells was assessed by fluorescent microscopy. Production of reactive oxygen species (ROS) by the cells was measured fluorometrically using Amplex Red. Production of tumour necrosis factor-alpha (TNF-α) by the colonic epithelial cells was analysed by ELISA. Nox1 gene expression was assessed by real-time PCR. RESULTS Our study showed that TNF-α level was increased in unstimulated primary colonic cells both in the acute and chronic colitis groups, whereas decreased viability, increased ROS production, and expression of Nox1 was characteristic only for chronic DSS colitis mice when compared to the controls. The stimulation by LPS increased ROS generation via NADPH oxidase and decreased cell viability in mice with acute colitis. Treatment with NADPH oxidase inhibitors increased cell viability and decreased the levels of ROS and TNF-α in the LPS-treated cells isolated from mice of both acute and chronic colitis groups. CONCLUSION Our study revealed the importance of NADPH oxidase in the pathogenesis of both acute and chronic inflammation of the colon.

TPMT and ITPA genetic variants in Lithuanian inflammatory bowel disease patients: Prevalence and azathioprine-related side effects

PURPOSE Inter-individual thiopurine metabolism variability can influence treatment outcomes in inflammatory bowel disease (IBD) patients. Genetic polymorphisms in thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphatase (ITPA) were linked with toxicity of azathioprine (AZA). The aim of the study was to investigate frequencies of TPMT and ITPA polymorphisms in Lithuanian IBD patients and analyze their association with AZA-related adverse events. MATERIALS/METHODS Polymorphisms in TPMT (TPMT*2,*3B,*3C,*3A) and ITPA (rs1127354, rs7270101) genes were determined using PCR-RFLP and TaqMan(®) genotyping assays. 551 consecutive Lithuanian IBD patients were genotyped. The use of AZA and its side effects were assessed retrospectively according to hospital medical records. RESULTS Frequencies of TPMT*3A, TPMT*3B and TPMT*3C alleles were 3.1%, 0.5% and 0.1%, respectively. TPMT*2 genetic variant was not detected in the study group. The distribution of minor alleles for ITPA rs1127354 and rs7270101 polymorphisms was 9.9% and 10.5%, respectively. AZA was prescribed in 82 patients and it provoked myelotoxicity in 11%, hepatotoxicity in 6.1%, dyspepsia in 6.1%, and pancreatitis in 3.6% of cases. Among patients who had AZA-related myelotoxicity, 11.1% were TPMT compound heterozygous, 44.4% had heterozygous genotype (P<0.01). Frequencies of ITPA minor alleles were similar among the patients with and without AZA-related side effects. CONCLUSION Frequencies of TPMT and ITPA variant alleles in Lithuanian IBD group were similar to those observed in the Northern-Eastern Europe Caucasian populations. Polymorphisms in TPMT might be associated with myelotoxicity and leukopenia in AZA treated patients, while ITPA variant alleles appear not to be linked with treatment-related side effects.

Replication study of ulcerative colitis risk loci in a Lithuanian-Latvian case-control sample

Background:Differences between populations might be reflected in their different genetic risk maps to complex diseases, for example, inflammatory bowel disease. We here investigated the role of known inflammatory bowel disease–associated single nucleotide polymorphisms (SNPs) in a subset of patients with ulcerative colitis (UC) from the Northeastern European countries Lithuania and Latvia and evaluated possible epistatic interactions between these genetic variants. Methods:We investigated 77 SNPs derived from 5 previously published genome-wide association studies for Crohns disease and UC. Our study panel comprised 444 Lithuanian and Latvian patients with UC and 1154 healthy controls. Single marker case–control association and SNP-SNP epistasis analyses were performed. Results:We found 14 SNPs tagging 9 loci, including 21q21.1, NKX2-3, MST1, the HLA region, 1p36.13, IL10, JAK2, ORMDL3, and IL23R, to be associated with UC. Interestingly, the association of UC with previously identified variants in the HLA region was not the strongest association in our study (P = 4.34 × 10−3, odds ratio [OR] = 1.25), which is in contrast to all previously published studies. No association with any disease subphenotype was found. SNP-SNP interaction analysis showed significant epistasis between SNPs in the PTPN22 (rs2476601) and C13orf31 (rs3764147) genes and increased risk for UC (P = 1.64 × 10−6, OR = 2.44). The association has been confirmed in the Danish study group (P = 0.04, OR = 3.25). Conclusions:We confirmed the association of the 9 loci (21q21.1, 1p36.13, NKX2-3, MST1, the HLA region, IL10, JAK2, ORMDL3, and IL23R) with UC in the Lithuanian–Latvian population. SNP-SNP interaction analyses showed that the combination of SNPs in the PTPN22 (rs2476601) and C13orf31 (rs3764147) genes increase the risk for UC.