Begoña Diosdado

Myosin IXB variant increases the risk of celiac disease and points toward a primary intestinal barrier defect

Celiac disease is probably the best-understood immune-related disorder. The disease presents in the small intestine and results from the interplay between multiple genes and gluten, the triggering environmental factor. Although HLA class II genes explain 40% of the heritable risk, non-HLA genes accounting for most of the familial clustering have not yet been identified. Here we report significant and replicable association (P = 2.1 × 10−6) to a common variant located in intron 28 of the gene myosin IXB (MYO9B), which encodes an unconventional myosin molecule that has a role in actin remodeling of epithelial enterocytes. Individuals homozygous with respect to the at-risk allele have a 2.3-times higher risk of celiac disease (P = 1.55 × 10−5). This result is suggestive of a primary impairment of the intestinal barrier in the etiology of celiac disease, which may explain why immunogenic gluten peptides are able to pass through the epithelial barrier.

MiR-17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression

Background:MicroRNAs are small non-coding RNA molecules, which regulate central mechanisms of tumorigenesis. In colorectal tumours, the combination of gain of 8q and 13q is one of the major factors associated with colorectal adenoma to adenocarcinoma progression. Functional studies on the miR-17-92 cluster localised on 13q31 have shown that its transcription is activated by c-myc, located on 8q, and that it has oncogenic activities. We investigated the contribution of the miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression.Methods:Expression levels of the miR-17-92 cluster were determined in 55 colorectal tumours and in 10 controls by real-time RT–PCR. Messenger RNA c-myc expression was also determined by real-time RT–PCR in 48 tumours with array comparative genomic hybridisation (aCGH) data available.Results:From the six members of the miR-17-92 cluster, all except miR-18a, showed significant increased expression in colorectal tumours with miR-17-92 locus gain compared with tumours without miR-17-92 locus gain. Unsupervised cluster analysis clustered the tumours based on the presence of miR-17-92 locus gain. Significant correlation between the expression of c-myc and the six miRNAs was also found.Conclusion:Increased expression of miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression is associated to DNA copy number gain of miR17-92 locus on 13q31 and c-myc expression.

A microarray screen for novel candidate genes in coeliac disease pathogenesis

Background and aims: The causative molecular pathways underlying the pathogenesis of coeliac disease are poorly understood. To unravel novel aspects of disease pathogenesis, we used microarrays to determine changes in gene expression of duodenal biopsies. Methods: cDNA microarrays representing 19 200 genes were used to compare gene expression profiles of duodenal biopsies from 15 coeliac disease patients with villous atrophy (Marsh III) and seven control individuals with normal biopsies (Marsh 0). In addition, the specific effect of gluten was studied by comparing the expression profiles of Marsh III lesions of seven patients exposed to gluten with four patients on a gluten free diet. Results: Comparing Marsh III with Marsh 0 lesions identified 109 genes that differed significantly (p<0.001) in expression levels between patients and controls. A large number of these genes have functions in proliferation and differentiation pathways and might be important for correct development of crypt-villous units. Alterations in these pathways may lead to the characteristic hyperplasia and villous atrophy seen in coeliac disease. The analyses also revealed 120 differentially expressed genes (p<0.005) when comparing patients on a gluten free diet with those exposed to gluten. These genes further strengthen our observation of increased cell proliferation in the presence of gluten. Conclusions: Our study provides new candidate genes in the pathogenesis of coeliac disease. Based on our results, we hypothesise that villous atrophy in coeliac disease patients is due to failure in cell differentiation. These genes are involved in pathways not previously implicated in coeliac disease pathogenesis and they may provide new targets for therapy.

Genetic variants of RANTES are associated with serum RANTES level and protection for type 1 diabetes

RANTES (regulated on activation, normal T-cell expressed and secreted) is a T-helper type 1 (Th1) chemokine that promotes T-cell activation and proliferation. RANTES is genetically associated with asthma, sarcoidosis and multiple sclerosis. The concentration of RANTES is increased at inflammation sites in different autoimmune diseases. Type 1 diabetes (T1D) is a Th1-mediated disease with complex genetic predisposition. We tested RANTES as a candidate gene for association with T1D using three single-nucleotide polymorphism (SNP) variants (rs4251719, rs2306630 and rs2107538) to capture haplotype information. The minor alleles of all SNPs were transmitted less frequently to T1D offspring (transmission rates 37.3% (P=0.002), 38.7% (P=0.007) and 41.0% (P=0.01)) and were less frequently present in patients compared to controls (P=0.009, 0.03 and 0.04, respectively). A similar protective effect was observed for the haplotype carrying three minor alleles (transmission disequilibrium test (TDT): P=0.003; odds ratio (OR)=0.55; confidence interval (CI): 0.37–0.83; case/control: P=0.03; OR=0.74; CI: 0.55–0.98). Both patients and controls carrying the protective haplotype express significantly lower serum levels of RANTES compared to non-carriers. Subsequently, we tested a cohort of 310 celiac disease patients, but failed to detect association. RANTES SNPs are significantly associated with RANTES serum concentration and development of T1D. The rs4251719*A–rs2306630*A–rs2107538*A haplotype associated with low RANTES production confers protection from T1D. Our data imply that RANTES is associated with T1D both genetically and functionally, and contributes to diabetes-prone Th1 cytokine profile.

High-Resolution Array Comparative Genomic Hybridization in Sporadic and Celiac Disease–Related Small Bowel Adenocarcinomas

Purpose: The molecular pathogenesis of small intestinal adenocarcinomas is not well understood. Understanding the molecular characteristics of small bowel adenocarcinoma may lead to more effective patient treatment. Experimental Design: Forty-eight small bowel adenocarcinomas (33 non–celiac disease related and 15 celiac disease related) were characterized for chromosomal aberrations by high-resolution array comparative hybridization, microsatellite instability, and APC promoter methylation and mutation status. Findings were compared with clinicopathologic and survival data. Furthermore, molecular alterations were compared between celiac disease–related and non–celiac disease–related small bowel adenocarcinomas. Results: DNA copy number changes were observed in 77% small bowel adenocarcinomas. The most frequent DNA copy number changes found were gains on 5p15.33-5p12, 7p22.3-7q11.21, 7q21.2-7q21.3, 7q22.1-7q34, 7q36.1, 7q36.3, 8q11.21-8q24.3, 9q34.11-9q34.3, 13q11-13q34, 16p13.3, 16p11.2, 19q13.2, and 20p13-20q13.33, and losses on 4p13-4q35.2, 5q15-5q21.1, and 21p11.2-21q22.11. Seven highly amplified regions were identified on 6p21.1, 7q21.1, 8p23.1, 11p13, 16p11.2, 17q12-q21.1, and 19q13.2. Celiac disease–related and non–celiac disease–related small bowel adenocarcinomas displayed similar chromosomal aberrations. Promoter hypermethylation of the APC gene was found in 48% non–celiac disease–related and 73% celiac disease–related small bowel adenocarcinomas. No nonsense mutations were found. Thirty-three percent of non–celiac disease–related small bowel adenocarcinomas showed microsatellite instability, whereas 67% of celiac disease–related small bowel adenocarcinomas were microsatellite unstable. Conclusions: Our study characterized chromosomal aberrations and amplifications involved in small bowel adenocarcinoma. At the chromosomal level, celiac disease–related and non–celiac disease–related small bowel adenocarcinomas did not differ. A defect in the mismatch repair pathways seems to be more common in celiac disease–related than in non–celiac disease–related small bowel adenocarcinomas. In contrast to colon and gastric cancers, no APC nonsense mutations were found in small bowel adenocarcinoma. However, APC promoter methylation seems to be a common event in celiac disease–related small bowel adenocarcinoma. Clin Cancer Res; 16(5); 1391–401

MLPAnalyzer: data analysis tool for reliable automated normalization of MLPA fragment data

Background: Multiplex Ligation dependent Probe Amplification (MLPA) is a rapid, simple, reliable and customized method for detection of copy number changes of individual genes at a high resolution and allows for high throughput analysis. This technique is typically applied for studying specific genes in large sample series. The large amount of data, dissimilarities in PCR efficiency among the different probe amplification products, and sample-to-sample variation pose a challenge to data analysis and interpretation. We therefore set out to develop an MLPA data analysis strategy and tool that is simple to use, while still taking into account the above-mentioned sources of variation. Materials and Methods: MLPAnalyzer was developed in Visual Basic for Applications, and can accept a large number of file formats directly from capillary sequence systems. Sizes of all MLPA probe signals are determined and filtered, quality control steps are performed, and variation in peak intensity related to size is corrected for. DNA copy number ratios of test samples are computed, displayed in a table view and a set of comprehensive figures is generated. To validate this approach, MLPA reactions were performed using a dedicated MLPA mix on 6 different colorectal cancer cell lines. The generated data were normalized using our program and results were compared to previously performed array-CGH results using both statistical methods and visual examination. Results and Discussion: Visual examination of bar graphs and direct ratios for both techniques showed very similar results, while the average Pearson moment correlation over all MLPA probes was found to be 0.42. Our results thus show that automated MLPA data processing following our suggested strategy may be of significant use, especially when handling large MLPA data sets, when samples are of different quality, or interpretation of MLPA electropherograms is too complex. It remains, however, important to recognize that automated MLPA data processing may only be successful when a dedicated experimental setup is also considered.

Coelionomics: towards understanding the molecular pathology of coeliac disease.

Abstract Coeliac disease (CD) is an inflammatory disorder of the small intestine characterised by a permanent intolerance to gluten-derived peptides. When gluten-derived peptides reach the lamina propria in CD patients, they provoke specific changes in the mucosa of their small intestine. Although the susceptibility to CD is strongly determined by environmental gluten, it is clearly a common genetic disorder. Important genetic factors for CD are the HLA-DQ genes located in the MHC region on chromosome 6 [ HLA-DQ2 (95%) or HLA-DQ8 (∼5%) heterodimers]. So far, the only treatment for CD consists of a life-long gluten-free diet. A key question in CD is why the gluten-derived peptides are resistant to further breakdown by endogenous proteases and how, in turn, they can activate a harmful immune response in the lamina propria of genetically predisposed individuals. Four mechanisms, namely apoptosis, oxidative stress, matrix metalloproteinases and dysregulation of proliferation and differentiation, are thought to play a role in the pathophysiology of CD. Whether the genes involved in these four mechanisms play a causative role in the development of the villous atrophy or are, in fact, a consequence of the disease process is unknown. In this review we summarise these mechanisms and discuss their validity in the context of current insights derived from genetic, genomic and molecular studies. We also discuss future directions for research and the therapeutic implications for patients.

TEAM: a tool for the integration of expression, and linkage and association maps

The identification of genes primarily responsible for complex genetic disorders is a daunting task. Despite the assignment of many susceptibility loci, there has only been limited success in identifying disease genes based solely on positional information from genome-wide screens. The incorporation of several complementary strategies in a single integrated approach should facilitate and further enhance the efficacy of this search for genes. To permit the integration of linkage, association and expression data, together with functional annotations, we have developed a Java-based software tool: TEAM (tool for the integration of expression, and linkage and association maps). TEAM includes a genome viewer, capable of overlaying karyobands, genes, markers, linkage graphs, association data, gene expression levels and functional annotations in one composite view. Data management, analysis and filtering functionality was implemented and extended with links to the Ensembl, Unigene and Gene Ontology databases to facilitate gene annotation. Filtering functionality can help prevent the exclusion of poorly annotated, but differentially expressed, genes that reside in candidate regions that show linkage or association. Here we demonstrate the programs functionality in our study on coeliac disease (OMIM 212750), a multifactorial gluten-sensitive enteropathy. We performed a combined data analysis of a genome-wide linkage screen in 82 Dutch families with affected siblings and the microarray expression profiles of 18 110 cDNAs in 22 intestinal biopsies.

Glucose Transporter 1 (SLC2A1) and Vascular Endothelial Growth Factor A (VEGFA) Predict Survival After Resection of Colorectal Cancer Liver Metastasis.

OBJECTIVE To investigate the individual and combined prognostic value of HIF1α, SLC2A1, and vascular endothelial growth factor A (VEGFA) in a multi-institutional cohort of patients with resected colorectal cancer liver metastasis (CRCLM). BACKGROUND In the majority of patients with CRCLM, resection seems not to be curative, despite its curative intent. Overexpression of hypoxia-inducible factor 1α (HIF1α), glucose transporter 1 (SLC2A1; also known as GLUT1), and VEGFA has been associated with tumor progression and poor prognosis of patients with colorectal cancer (CRC). METHODS Tissue microarrays were generated using CRCLM and patient-matched primary CRC from patients who underwent CRCLM resection between 1990 and 2010. Prognostic value of HIF1α, SLC2A1, and VEGFA was determined by immunohistochemistry. A 500-fold cross-validated hazard rate ratio (HRRav) for overall survival was calculated. RESULTS HIF1α, SLC2A1, and VEGFA expression could be evaluated in 328, 350, and 335 patients, respectively. High SLC2A1 expression was associated with good prognosis (HRRav, 0.67; P (HRR >1)  < 0.01) and high VEGFA expression to poor prognosis (HRRav, 1.84; P (HRR < 1)  = 0.02), also after multivariate analysis including established clinicopathological prognostic variables (HRRav, 0.67; P (HRR > 1)  < 0.01 and HRRav, 1.50; P (HRR < 1)  = 0.02, respectively). SLC2A1 showed prognostic value particularly in patients treated with systemic therapy (P < 0.01), whereas the prognostic value of VEGFA expression was mainly observed in patients not treated with systemic therapy (P < 0.01). Prognosis was especially poor in patients with both low SLC2A1 and high VEGFA expression (P < 0.01). HIF1α expression was not associated with survival. CONCLUSIONS SLC2A1 and VEGFA expression are prognostic molecular biomarkers for patients with CRCLM with added value to established clinicopathological variables.

Molecular mechanisms of the adaptive, innate and regulatory immune responses in the intestinal mucosa of celiac disease patients.

Celiac disease is a complex genetic disorder that affects the small intestine of genetically predisposed individuals when they ingest gluten, a dietary protein. Although several genome screens have been successful in identifying susceptibility loci in celiac disease, the only genetic contributors identified so far are the human leukocyte antigen (HLA)-DQ2/DQ8 molecules. One of the most important aspects in the pathogenesis of celiac disease is the activation of a T-helper 1 immune response, when the antigen-presenting cells that express HLA-DQ2/DQ8 molecules present the toxic gluten peptides to reactive CD4+ T-cells. Recently, new insights into the activation of an innate immune response have also been described. It is generally accepted that the immune response triggers destruction of the mucosa in the small intestine of celiac disease patients. Hence, the activation of a detrimental immune response in the intestine of celiac disease patients appears to be key in the initiation and progression of the disease. This review summarizes the immunologic pathways that have been studied in celiac disease thus far, and will point to new potential candidate genes and pathways involved in the etiopathogenesis of celiac disease, which should lead to novel alternatives for diagnosis and treatment.