Circular RNAs Could Encode Unique Proteins and Affect Cancer Pathways.

circRNAs constitute a novel class of RNA, generally considered as non-coding RNAs; nonetheless, their coding potential has been under scrutiny. In this work, we systematically explored the predicted proteins of more than 160,000 circRNAs detected by exome capture RNA-sequencing and collected in the MiOncoCirc pan-cancer compendium, including normal and cancer samples from different types of tissues. For the functional evaluation, we compared their primary structure and domain composition with those derived from the same linear mRNAs. Among the 4362 circRNAs potentially encoding proteins with a unique primary structure and 1179 encoding proteins with a novel domain composition, 183 were differentially expressed in cancer. In particular, eight were associated with prognosis in acute myeloid leukemia. The functional classification of the dysregulated circRNA-encoded polypeptides showed an enrichment in the heme and cancer signaling, DNA-binding, and phosphorylation processes, and disclosed the roles of some circRNA-based effectors in cancer.

Cereblon (CRBN) gene polymorphisms predict clinical response and progression-free survival in relapsed/refractory multiple myeloma patients treated with lenalidomide: a pharmacogenetic study from the IMMEnSE consortium.

Cereblon (CRBN) is crucial for antiproliferative and immunomodulatory properties of immunomodulatory drugs. The objective of this study was to verify whether germline single nucleotide polymorphisms (SNPs) in the CRBN gene may influence response to lenalidomide in multiple myeloma (MM). Fourteen tagging SNPs covering the genetic variability in the CRBN gene region were genotyped in 167 Polish patients with refractory/relapsed MM treated with lenalidomide-based regimens. We found that carriers of minor alleles of two studied CRBN SNPs rs1714327G > C (OR = 0.26; 95% CI = 0.1-0.67; p = .0055, Bonferroni corrected p = .033) and rs1705814T > C (OR = 0.22; 95% CI = 0.07-0.65; p = .0063, Bonferroni corrected p = .037) were significantly associated with lower probability of achievement at least partial remission while treated with lenalidomide-based regimens, using the dominant inheritance model. Moreover, one of these SNPs, namely rs1705814T > C, was correlated with shorter progression-free survival (HR = 2.49; 95%CI = 1.31-4.74, p = .0054, Bonferroni corrected p = .033). It is suggested that selected germline CRBN allelic variants (rs1714327G > C and rs1705814T > C) affect lenalidomide efficacy in patients with relapsed/refractory MM.

Small extracellular vesicles containing arginase-1 suppress T-cell responses and promote tumor growth in ovarian carcinoma.

Tumor-driven immune suppression is a major barrier to successful immunotherapy in ovarian carcinomas (OvCa). Among various mechanisms responsible for immune suppression, arginase-1 (ARG1)-carrying small extracellular vesicles (EVs) emerge as important contributors to tumor growth and tumor escape from the host immune system. Here, we report that small EVs found in the ascites and plasma of OvCa patients contain ARG1. EVs suppress proliferation of CD4+ and CD8+ T-cells in vitro and in vivo in OvCa mouse models. In mice, ARG1-containing EVs are transported to draining lymph nodes, taken up by dendritic cells and inhibit antigen-specific T-cell proliferation. Increased expression of ARG1 in mouse OvCa cells is associated with accelerated tumor progression that can be blocked by an arginase inhibitor. Altogether, our studies show that tumor cells use EVs as vehicles to carry over long distances and deliver to immune cells a metabolic checkpoint molecule - ARG1, mitigating anti-tumor immune responses.

Peroxiredoxin-5 is a negative survival predictor in ovarian cancer.

OBJECTIVES: Peroxiredoxins (PRDXs) constitute a family of antioxidant enzymes which are also involved in the process of carcinogenesis. They are composed of six identified isoforms (PRDX-1-6) and are supposed to play different roles in tumor progression, depending on type of cancer and member of the PRDX family. The aim of the study was to assess the prog- nostic value of PRDXs in ovarian cancer. MATERIAL AND METHODS: a dataset of patients with ovarian cancer from The Cancer Genome Atlas was analyzed. Expression of PRDX-1 to 6 mRNA was evaluated in 260 samples. The prognostic value of PRDXs was assessed using the Cox regression model which included the following clinical and pathological data: age, clinical stage, tumor grade, and residual disease. RESULTS: Within the PRDXs family, only higher expression of PRDX-5 was associated with worse overall survival both, in unselected patients and > 50-year-olds. PRDX-5 expression and residual disease were independent negative prognostic factors of patient survival. CONCLUSIONS: PRDX-5 is a negative predictor of survival in ovarian cancer.

Targeting peroxiredoxin 1 impairs growth of breast cancer cells and potently sensitises these cells to prooxidant agents.

BACKGROUND: Our previous work has shown peroxiredoxin-1 (PRDX1), one of major antioxidant enzymes, to be a biomarker in human breast cancer. Hereby, we further investigate the role of PRDX1, compared to its close homolog PRDX2, in mammary malignant cells. METHODS: CRISPR/Cas9- or RNAi-based methods were used for genetic targeting PRDX1/2. Cell growth was assessed by crystal violet, EdU incorporation or colony formation assays. In vivo growth was assessed by a xenotransplantation model. Adenanthin was used to inhibit the thioredoxin-dependent antioxidant defense system. The prooxidant agents used were hydrogen peroxide, glucose oxidase and sodium L-ascorbate. A PY1 probe or HyPer-3 biosensor were used to detect hydrogen peroxide content in samples. RESULTS: PRDX1 downregulation significantly impaired the growth rate of MCF-7 and ZR-75-1 breast cancer cells. Likewise, xenotransplanted PRDX1-deficient MCF-7 cells presented a retarded tumour growth. Furthermore, genetic targeting of PRDX1 or adenanthin, but not PRDX2, potently sensitised all six cancer cell lines studied, but not the non-cancerous cells, to glucose oxidase and ascorbate. CONCLUSIONS: Our study pinpoints the dominant role for PRDX1 in management of exogeneous oxidative stress by breast cancer cells and substantiates further exploration of PRDX1 as a target in this disease, especially when combined with prooxidant agents.

Inhibition of protein disulfide isomerase induces differentiation of acute myeloid leukemia cells.

A cute myeloid leukemia is a malignant disease of immature myeloid cells. Despite significant therapeutic effects of differentiation-inducing agents in some acute myeloid leukemia subtypes, the disease remains incurable in a large fraction of patients. Here we show that SK053, a thioredoxin inhibitor, induces differentiation and cell death of acute myeloid leukemia cells. Considering that thioredoxin knock-down with short hairpin RNA failed to exert antiproliferative effects in one of the acute myeloid leukemia cell lines, we used a biotin affinity probe-labeling approach to identify potential molecular targets for the effects of SK053. Mass spectrometry of proteins precipitated from acute myeloid leukemia cells incubated with biotinylated SK053 used as a bait revealed protein disulfide isomerase as a potential binding partner for the compound. Biochemical, enzymatic and functional assays using fluorescence lifetime imaging confirmed that SK053 binds to and inhibits the activity of protein disulfide isomerase. Protein disulfide isomerase knockdown with short hairpin RNA was associated with inhibition of cell growth, increased CCAAT enhancer-binding protein α levels, and induction of differentiation of HL-60 cells. Molecular dynamics simulation followed by the covalent docking indicated that SK053 binds to the fourth thioredoxin-like domain of protein disulfide isomerase. Differentiation of myeloid precursor cells requires the activity of CCAAT enhancer-binding protein α, the function of which is impaired in acute myeloid leukemia cells through various mechanisms, including translational block by protein disulfide isomerase. SK053 increased the levels of CCAAT enhancer-binding protein α and upregulated mRNA levels for differentiation-associated genes. Finally, SK053 decreased the survival of blasts and increased the percentage of cells expressing the maturation-associated CD11b marker in primary cells isolated from bone marrow or peripheral blood of patients with acute myeloid leukemia. Collectively, these results provide a proof-of-concept that protein disulfide isomerase inhibition has potential as a therapeutic strategy for the treatment of acute myeloid leukemia and for the development of small-molecule inhibitors of protein disulfide isomerase.

Functional analysis of a novel, thyroglobulin-embedded microRNA gene deregulated in papillary thyroid carcinoma.

MicroRNAs, non-coding regulators of gene expression, are known culprits of thyroid cancer. Using next-generation sequencing, we identified a novel microRNA gene, encoded within an important thyroid regulator - thyroglobulin, and analyzed its functionality in the thyroid gland. In vitro and in silico analyses proved that the novel miR-TG is processed from the precursor, and co-expressed with thyroglobulin. Both genes are specific for thyroid tissue and downregulated in papillary thyroid carcinoma by 44% (p = 0.04) and 48% (p = 0.001), respectively. Putative target genes for miR-TG were identified using in silico tools, which pinpointed MAP4K4, an oncogene upregulated in thyroid cancer. Analysis of transcriptome by RNA-seq revealed that overexpression of miR-TG in PTC-derived cell line led to downregulation of several genes, including MAP4K4 (fold change 0,82; p = 0.036). The finding was confirmed by SQ-PCR (fold change 071; p = 0.004). Direct interaction between miR-TG and MAP4K4 was confirmed in the luciferase assay (p = 0.0006). Functional studies showed increase proliferation in K1 cell line transfected with miR-TG. We propose that in normal thyroid miR-TG plays a fine-tuning effect on the maintenance of MAPK pathway, inhibiting the expression of miR's target MAP4K4. This regulation is disturbed in cancer due to downregulation of the novel, thyroglobulin-embedded microRNA, characterized in this study.

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma.

Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease.

Peroxiredoxin-1 protects estrogen receptor α from oxidative stress-induced suppression and is a protein biomarker of favorable prognosis in breast cancer.

INTRODUCTION: Peroxiredoxin-1 (PRDX1) is a multifunctional protein, acting as a hydrogen peroxide (H2O2) scavenger, molecular chaperone and immune modulator. Although differential PRDX1 expression has been described in many tumors, the potential role of PRDX1 in breast cancer remains highly ambiguous. Using a comprehensive antibody-based proteomics approach, we interrogated PRDX1 protein as a putative biomarker in estrogen receptor (ER)-positive breast cancer. METHODS: An anti-PRDX1 antibody was validated in breast cancer cell lines using immunoblotting, immunohistochemistry and reverse phase protein array (RPPA) technology. PRDX1 protein expression was evaluated in two independent breast cancer cohorts, represented on a screening RPPA (n = 712) and a validation tissue microarray (n = 498). In vitro assays were performed exploring the functional contribution of PRDX1, with oxidative stress conditions mimicked via treatment with H2O2, peroxynitrite, or adenanthin, a PRDX1/2 inhibitor. RESULTS: In ER-positive cases, high PRDX1 protein expression is a biomarker of improved prognosis across both cohorts. In the validation cohort, high PRDX1 expression was an independent predictor of improved relapse-free survival (hazard ratio (HR) = 0.62, 95% confidence interval (CI) = 0.40 to 0.96, P = 0.032), breast cancer-specific survival (HR = 0.44, 95% CI = 0.24 to 0.79, P = 0.006) and overall survival (HR = 0.61, 95% CI = 0.44 to 0.85, P = 0.004). RPPA screening of cancer signaling proteins showed that ERα protein was upregulated in PRDX1 high tumors. Exogenous H2O2 treatment decreased ERα protein levels in ER-positive cells. PRDX1 knockdown further sensitized cells to H2O2- and peroxynitrite-mediated effects, whilst PRDX1 overexpression protected against this response. Inhibition of PRDX1/2 antioxidant activity with adenanthin dramatically reduced ERα levels in breast cancer cells. CONCLUSIONS: PRDX1 is shown to be an independent predictor of improved outcomes in ER-positive breast cancer. Through its antioxidant function, PRDX1 may prevent oxidative stress-mediated ERα loss, thereby potentially contributing to maintenance of an ER-positive phenotype in mammary tumors. These results for the first time imply a close connection between biological activity of PRDX1 and regulation of estrogen-mediated signaling in breast cancer.

Modulation of age- and cancer-associated DNA methylation change in the healthy colon by aspirin and lifestyle.

BACKGROUND: Aberrant DNA methylation in gene promoters is associated with aging and cancer, but the circumstances determining methylation change are unknown. We investigated the impact of lifestyle modulators of colorectal cancer (CRC) risk on the stability of gene promoter methylation in the colonic mucosa. METHODS: We measured genome-wide promoter CpG methylation in normal colon biopsies (n = 1092) from a female screening cohort, investigated the interaction of lifestyle factors with age-dependent increase in methylation with log-linear multivariable regression, and related their modifying effect to hypermethylation in CRC. All statistical tests were two-sided. RESULTS: Of 20025 promoter-associated CpGs analyzed, 1713 showed statistically significant age-dependent methylation gains. Fewer CpGs acquired methylation in users of aspirin (≥ 2 years) and hormonal replacement therapy (HRT age ≥ 50 years) compared with nonusers (43 vs 1355; 1 vs1377, respectively), whereas more CpGs were affected in smokers (≥ 20 years) and individuals with a body mass index (BMI) of 25 kg/m(2) and greater compared with control groups (180 vs 39; 554 vs 144, respectively). Fifty percent of the CpGs showing age-dependent methylation were found hypermethylated in CRC (odds ratio [OR] = 20; 95% confidence interval [CI] = 18 to 23; P < 2 × 10(-16)). These loci gained methylation with a higher median rate compared with age-only methylated sites (P = 2 × 10(-76)) and were enriched for polycomb regions (OR = 3.67). Importantly, aspirin (P < .001) and HRT use (P < .001) reduced the methylation rate at these cancer-related genes, whereas smoking (P < .001) and high BMI (P = .004) increased it. CONCLUSIONS: Lifestyle, including aspirin use, modulates age-associated DNA methylation change in the colonic epithelium and thereby impacts the evolution of cancer methylomes.

CYP1A1 Ile462Val polymorphism and colorectal cancer risk in Polish patients.

Colorectal cancer (CRC) is an epidemiological problem of a great importance in Poland; each year approximately 14,600 new cases of the disease are diagnosed. Mortality associated with CRC reaches approximately 10,400 cases per year (according to the National Cancer Registry). The 5-year survival rate is approximately 25 %, which is one of the lowest rates in Europe. The etiology of sporadic colorectal cancer (CRC) is multifactorial and has been attributed to an interplay between both environmental and genetic risk factors. In addition, there is a general consensus that genetic factors may modulate the influence of environmental insults. Following these assumptions, we performed a study on widely described polymorphisms in xenobiotic-metabolizing enzymes and DNA repair genes which may influence individual susceptibility to cancer. We selected five candidate polymorphisms in following genes: ERCC1 Asp118Asn (rs11615), XPC i11C/A (rs2279017), XRCC3 Met241Thr (rs861539) CYP1A1 Ile462Val (rs1048943) and NAT2 A803G (rs1208) and assessed the importance of chosen SNPs on groups consisting of 478 CRC patients and 404 controls. Only CYP1A1 Ile462Val was statistically significant in CRC patients over 50 years old: OR 2.05 (1.29-3.28); p = 1.25E-02 and this association was more pronounced in the female group of CRC patients after the age of 50: OR 2.72 (1.43-5.14); p = 1.14E-02.

Association of the BRCA1 promoter polymorphism rs11655505 with the risk of familial breast and/or ovarian cancer.

Germline mutations in the BRCA1 tumor suppressor gene predispose affected individuals to breast cancer; however, incomplete cancer penetrance and the presence of phenocopies in BRCA1 families also indicate genetic and environmental modifiers of breast cancer risk. In this study, we have tested the single nucleotide polymorphism rs1655505 of the BRCA1 promoter, as candidate for the modifier of breast cancer risk. The polymorphic variants were genotyped in BRCA1-negative (729), familial breast and/or ovarian cancer cases (FBOC), including cases with a reported maternal history (154), nonfamilal (sporadic) cases (600), hereditary breast/ovarian cases with BRCA1 mutations (190) and population controls (1,590) from Central Poland. An association with the risk of FBOC was observed for the minor (T) allele and (TT) genotype (T: p = 0.006, OR = 1.40, 95% CI = 1.10-1.79; TT: p = 0.001, OR = 2.23, 95% CI = 1.37-3.62) in female cases with a reported maternal history, specifically in women with the onset of disease after 50 years of age (T: p = 0.004, OR = 1.77, 95% CI = 1.20-2.62; TT: p = 0.001, OR = 3.7, 95% CI = 1.62-8.46). The presented evidence suggests a need to conduct larger studies on the association between genetic variations at the BRCA1 promoter and the breast cancer risk, according to maternal/paternal lineage.

Association of genetic variation in the NR1H4 gene, encoding the nuclear bile acid receptor FXR, with inflammatory bowel disease.

BACKGROUND: Pathogenesis of inflammatory bowel diseases (IBD), ulcerative colitis (UC) and Crohn's disease (CD), involves interaction between environmental factors and inappropriate immune responses in the intestine of genetically predisposed individuals. Bile acids and their nuclear receptor, FXR, regulate inflammatory responses and barrier function in the intestinal tract. METHODS: We studied the association of five variants (rs3863377, rs7138843, rs56163822, rs35724, rs10860603) of the NR1H4 gene encoding FXR with IBD. 1138 individuals (591 non-IBD, 203 UC, 344 CD) were genotyped for five NR1H4 genetic variants with TaqMan SNP Genotyping Assays. RESULTS: We observed that the NR1H4 SNP rs3863377 is significantly less frequent in IBD cases than in non-IBD controls (allele frequencies: P = 0.004; wild-type vs. SNP carrier genotype frequencies: P = 0.008), whereas the variant rs56163822 is less prevalent in non-IBD controls (allele frequencies: P = 0.027; wild-type vs. SNP carrier genotype frequencies: P = 0.035). The global haplotype distribution between IBD and control patients was significantly different (P = 0.003). This also held true for the comparison between non-IBD and UC groups (P = 0.004), but not for the comparison between non-IBD and CD groups (P = 0.079). CONCLUSIONS: We show that genetic variation in FXR is associated with IBD, further emphasizing the link between bile acid signaling and intestinal inflammation.

High frequency of BRCA1 founder mutations in Polish women with nonfamilial breast cancer.

Possession of a BRCA1/2 mutation increases risk of contralateral breast and ovarian cancer recurrence and may have an impact on health management decisions, such as imaging screening, preventive surgical interventions and systemic therapies. A hospital-based study was conducted to assess the frequency and spectrum of pathogenic germline BRCA1 and BRCA2 mutations in Polish women with familial and nonfamilial breast cancer. Genomic DNA was extracted from 1581 women with breast cancer and from 2225 healthy individuals. For genotyping BRCA1 (5382insC, T300G, 3819del5, 185delAG, C5370T, 3875del4, 3896delT, 4153delA, 4184del4, 4160delAG, G5332A) mutations and BRCA2 (G1408T, 5467insT, 6174delT, 6192delAT, 6675delTA, 8138del5, 9152delT, C9610T, 9630delC) mutations, a Custom TaqMan (Applied Biosystems) PCR-based technology was adopted. A BRCA1 mutation was found in 26 and 12.5 % of women with familial breast cancer and in 13 and 8.3 % nonfamilial (sporadic) breast cancer, diagnosed before or after 50 years of age, respectively. A much lower frequency of BRCA2 mutation was observed. The predominance of seven BRCA1 mutations (5382insC, T300G, 3819del5, 185delAG, C5370T, 3875del4, 4153delA) studied in the Masovian voivodeship population confirmed a strong founder effect for BRCA1 mutations in the Polish population, and the results of BRCA2 testing confirmed a high diversity in the studied pathogenic mutations in BRCA2 gene. We propose offering inexpensive testing for the presence of BRCA1 founder mutations to all Polish women at the time of initial breast cancer diagnosis, regardless of the patient's family history or age of disease onset.

Pooled sample-based GWAS: a cost-effective alternative for identifying colorectal and prostate cancer risk variants in the Polish population.

BACKGROUND: Prostate cancer (PCa) and colorectal cancer (CRC) are the most commonly diagnosed cancers and cancer-related causes of death in Poland. To date, numerous single nucleotide polymorphisms (SNPs) associated with susceptibility to both cancer types have been identified, but their effect on disease risk may differ among populations. METHODS: To identify new SNPs associated with PCa and CRC in the Polish population, a genome-wide association study (GWAS) was performed using DNA sample pools on Affymetrix Genome-Wide Human SNP 6.0 arrays. A total of 135 PCa patients and 270 healthy men (PCa sub-study) and 525 patients with adenoma (AD), 630 patients with CRC and 690 controls (AD/CRC sub-study) were included in the analysis. Allele frequency distributions were compared with t-tests and χ(2)-tests. Only those significantly associated SNPs with a proxy SNP (p<0.001; distance of 100 kb; r(2)>0.7) were selected. GWAS marker selection was conducted using PLINK. The study was replicated using extended cohorts of patients and controls. The association with previously reported PCa and CRC susceptibility variants was also examined. Individual patients were genotyped using TaqMan SNP Genotyping Assays. RESULTS: The GWAS selected six and 24 new candidate SNPs associated with PCa and CRC susceptibility, respectively. In the replication study, 17 of these associations were confirmed as significant in additive model of inheritance. Seven of them remained significant after correction for multiple hypothesis testing. Additionally, 17 previously reported risk variants have been identified, five of which remained significant after correction. CONCLUSION: Pooled-DNA GWAS enabled the identification of new susceptibility loci for CRC in the Polish population. Previously reported CRC and PCa predisposition variants were also identified, validating the global nature of their associations. Further independent replication studies are required to confirm significance of the newly uncovered candidate susceptibility loci.

Crohn's disease-associated polymorphism within the PTPN2 gene affects muramyl-dipeptide-induced cytokine secretion and autophagy.

BACKGROUND: The single nucleotide polymorphism (SNP) rs2542151 within the gene locus region encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) has been associated with Crohn's disease (CD), ulcerative colitis (UC), type-I diabetes, and rheumatoid arthritis. We have previously shown that PTPN2 regulates mitogen-activated protein kinase (MAPK) signaling and cytokine secretion in human THP-1 monocytes and intestinal epithelial cells (IEC). Here, we studied whether intronic PTPN2 SNP rs1893217 regulates immune responses to the nucleotide-oligomerization domain 2 (NOD2) ligand, muramyl-dipeptide (MDP). MATERIALS AND METHODS: Genomic DNA samples from 343 CD and 663 non-IBD control patients (male and female) from a combined German, Swiss, and Polish cohort were genotyped for the presence of the PTPN2 SNPs, rs2542151, and rs1893217. PTPN2-variant rs1893217 was introduced into T(84) IEC or THP-1 cells using a lentiviral vector. RESULTS: We identified a novel association between the genetic variant, rs1893217, located in intron 7 of the PTPN2 gene and CD. Human THP-1 monocytes carrying this variant revealed increased MAPK activation as well as elevated mRNA expression of T-bet transcription factor and secretion of interferon-γ in response to the bacterial wall component, MDP. In contrast, secretion of interleukin-8 and tumor necrosis factor were reduced. In both, T(84) IEC and THP-1 monocytes, autophagosome formation was impaired. CONCLUSIONS: We identified a novel CD-associated PTPN2 variant that modulates innate immune responses to bacterial antigens. These findings not only provide key insights into the effects of a functional mutation on a clinically relevant gene, but also reveal how such a mutation could contribute to the onset of disease.

Lack of evidence for association of primary sclerosing cholangitis and primary biliary cirrhosis with risk alleles for Crohn's disease in Polish patients.

BACKGROUND: Numerous papers have addressed the association of mutations and polymorphisms of susceptibility genes with autoimmune inflammatory disorders. We investigated whether polymorphisms that confer susceptibility to Crohn's disease could be classified also as predisposing factors for the development of primary sclerosing cholangitis and primary biliary cirrhosis in Polish patients. METHODS: The study included 60 patients with CD, 77 patients with PSC, of which 61 exhibited IBD (40 UC, 8 CD, and 13 indeterminate colitis), and 144 patients with PBC. All the patients were screened against Crohn's disease associating genetic polymorphisms. The polymorphisms were chosen according to previously confirmed evidence for association with Crohn's disease, including Pro268Ser, Arg702Trp, Gly908Arg and 1007fs in NOD2/CARD15, Leu503Phe/-207G>C in SLC22A4/OCTN1/SLC22A5/OCTN2, Arg30Gln in DLG5, Thr300Ala in ATG16L1, and Arg381Gln, His3Gln and exon-3'UTR in IL23R. Genotyping was carried out using TaqMan SNP genotyping assays. RESULTS: We confirmed a strong association between three NOD2/CARD15 gene variants (Pro268Ser, OR = 2.52, 95% CI = 1.34-4.75); (Arg702Trp, OR = 6.65, 95% CI = 1.99-22.17); (1007fs, OR = 9.59, 95% CI = 3.94-23.29), and a weak association between both the protective OCTN1/OCTN2 CC haplotype (OR = 0.28, 95% CI = 0.08-0.94), and a variant of ATG16L1 gene (Thr300Ala, OR = 0.468, 95% CI = 0.24-0.90) with Crohn's disease. In contrast, none of the polymorphisms exhibited association with susceptibility to primary sclerosing cholangitis and primary biliary cirrhosis, including a group of primary sclerosing cholangitis patients with concurrent IBD. CONCLUSION: Although the clinical data indicate non-random co-occurrence of inflammatory bowel disease and primary sclerosing cholangitis, consistently with the previously published studies, no genetic association was found between the genetic variants predisposing to Crohn's disease and hepatobiliary autoimmune disorders. However, since estimation of genetic variant disproportion is limited by sample size, these negative results may also indicate that eventually shared genetic predispositions are too little to be captured by small patient groups.

Barrett's esophagus associates with a variant of IL23R gene.

Gastroesophageal reflux disease is regarded as a spectrum of diseases: non-erosive reflux disease (NERD), erosive reflux disease (ERD), and the far end of the spectrum represented by patients with Barrett's esophagus. Among predisposing factors, both risk and protective polymorphic variants of several genes may influence the clinical outcomes of reflux disease. Consequently, different molecular mechanisms are likely to underlie the development of clinical variants of reflux disease. Ninety six patients with reflux disease were screened for polymorphisms of CARD15, SLC22A4 (OCTN1), SLC22A5 (OCTN2), DLG5, ATG16L1 and IL23R genes which had previously been found to associate with immune-mediated chronic inflammatory disorders. While none of the polymorphisms were associated with NERD or ERD, the 1142G/A variant of the IL23R gene was found to be a risk variant in Barrett's esophagus patients. The IL23/IL23R pathway may modulate STAT3 transcriptional activity which is an essential regulator not only of immune-mediated inflammation, but also of inflammatory-associated apoptosis resistance. Although the mechanisms of metaplastic transition of inflamed squamous epithelium are undetermined as yet, our findings suggest potential involvement of alternations in the IL23/IL23R pathway as a molecular background of Barrett's esophagus development.

Molecular defense mechanisms of Barrett's metaplasia estimated by an integrative genomics.

Barrett's esophagus is characterized by the replacement of squamous epithelium with specialized intestinal metaplastic mucosa. The exact mechanisms of initiation and development of Barrett's metaplasia remain unknown, but a hypothesis of "successful adaptation" against noxious reflux components has been proposed. To search for the repertoire of adaptation mechanisms of Barrett's metaplasia, we employed high-throughput functional genomic and proteomic methods that defined the molecular background of metaplastic mucosa resistance to reflux. Transcriptional profiling was established for 23 pairs of esophageal squamous epithelium and Barrett's metaplasia tissue samples using Affymetrix U133A 2.0 GeneChips and validated by quantitative real-time polymerase chain reaction. Differences in protein composition were assessed by electrophoretic and mass-spectrometry-based methods. Among 2,822 genes differentially expressed between Barrett's metaplasia and squamous epithelium, we observed significantly overexpressed metaplastic mucosa genes that encode cytokines and growth factors, constituents of extracellular matrix, basement membrane and tight junctions, and proteins involved in prostaglandin and phosphoinositol metabolism, nitric oxide production, and bioenergetics. Their expression likely reflects defense and repair responses of metaplastic mucosa, whereas overexpression of genes encoding heat shock proteins and several protein kinases in squamous epithelium may reflect lower resistance of normal esophageal epithelium than Barrett's metaplasia to reflux components. Despite the methodological and interpretative difficulties in data analyses discussed in this paper, our studies confirm that Barrett's metaplasia may be regarded as a specific microevolution allowing for accumulation of mucosal morphological and physiological changes that better protect against reflux injury.