Increased Relative Abundance of Ruminoccocus Is Associated With Reduced Cardiovascular Risk in an Obese Population.

Background: Obesity is a complex disease with underlying genetic, environmental, psychological, physiological, medical, and epigenetic factors. Obesity can cause various disorders, including cardiovascular diseases (CVDs), that are among the most prevalent chronic conditions in Qatar. Recent studies have highlighted the significant roles of the gut microbiome in improving the pathology of various diseases, including obesity. Thus, in this study, we aimed to investigate the effects of dietary intake and gut microbial composition in modulating the risk of CVD development in obese Qatari adults. Methods: We enrolled 46 adult subjects (18-65 years of age) who were classified based on their CVD risk scores, calculated using the Framingham formula, into a CVD no-risk group (score of <10%, n = 36) and CVD risk group (score of ≥10%, n = 10). For each study subject, we measured the gut microbial composition with a 16s rDNA sequencing method that targeted the v3-v4 region using Illumina Miseq, and their nutritional status was recorded based on 24-h dietary recall. Dietary intake, bacterial taxa summary, diversity index, microbial markers, pathway analysis, and network correlation were determined for the study subjects. Results: The CVD risk group showed a lower intake of vitamin D, reduced relative abundance of genera Ruminococcus and Bifidobacterium, no change in bacterial diversity, and higher levels of taurine, hypotaurine, and lipoic acid metabolism than the CVD no-risk group. Besides, the relative abundance of genus Ruminococcus was positively correlated with the intake of protein, monounsaturated fat, vitamin A, and vitamin D. Conclusion: Taken together, our results suggest that the genus Ruminococcus could be used as a microbial marker, and its reduced relative abundance could mediate the risk of CVDs in the Obese Qatari population.

A population study of clinically actionable genetic variation affecting drug response from the Middle East.

Clinical implementation of pharmacogenomics will help in personalizing drug prescriptions and alleviate the personal and financial burden due to inefficacy and adverse reactions to drugs. However, such implementation is lagging in many parts of the world, including the Middle East, mainly due to the lack of data on the distribution of actionable pharmacogenomic variation in these ethnicities. We analyzed 6,045 whole genomes from the Qatari population for the distribution of allele frequencies of 2,629 variants in 1,026 genes known to affect 559 drugs or classes of drugs. We also performed a focused analysis of genotypes or diplotypes of 15 genes affecting 46 drugs, which have guidelines for clinical implementation and predicted their phenotypic impact. The allele frequencies of 1,320 variants in 703 genes affecting 299 drugs or class of drugs were significantly different between the Qatari population and other world populations. On average, Qataris carry 3.6 actionable genotypes/diplotypes, affecting 13 drugs with guidelines for clinical implementation, and 99.5% of the individuals had at least one clinically actionable genotype/diplotype. Increased risk of simvastatin-induced myopathy could be predicted in ~32% of Qataris from the diplotypes of SLCO1B1, which is higher compared to many other populations, while fewer Qataris may need tacrolimus dosage adjustments for achieving immunosuppression based on the CYP3A5 diplotypes compared to other world populations. Distinct distribution of actionable pharmacogenomic variation was also observed among the Qatari subpopulations. Our comprehensive study of the distribution of actionable genetic variation affecting drugs in a Middle Eastern population has potential implications for preemptive pharmacogenomic implementation in the region and beyond.

Genome sequencing data analysis for rare disease gene discovery.

Rare diseases occur in a smaller proportion of the general population, which is variedly defined as less than 200 000 individuals (US) or in less than 1 in 2000 individuals (Europe). Although rare, they collectively make up to approximately 7000 different disorders, with majority having a genetic origin, and affect roughly 300 million people globally. Most of the patients and their families undergo a long and frustrating diagnostic odyssey. However, advances in the field of genomics have started to facilitate the process of diagnosis, though it is hindered by the difficulty in genome data analysis and interpretation. A major impediment in diagnosis is in the understanding of the diverse approaches, tools and datasets available for variant prioritization, the most important step in the analysis of millions of variants to select a few potential variants. Here we present a review of the latest methodological developments and spectrum of tools available for rare disease genetic variant discovery and recommend appropriate data interpretation methods for variant prioritization. We have categorized the resources based on various steps of the variant interpretation workflow, starting from data processing, variant calling, annotation, filtration and finally prioritization, with a special emphasis on the last two steps. The methods discussed here pertain to elucidating the genetic basis of disease in individual patient cases via trio- or family-based analysis of the genome data. We advocate the use of a combination of tools and datasets and to follow multiple iterative approaches to elucidate the potential causative variant.

Can the Salivary Microbiome Predict Cardiovascular Diseases? Lessons Learned From the Qatari Population.

Background: Many studies have linked dysbiosis of the gut microbiome to the development of cardiovascular diseases (CVD). However, studies assessing the association between the salivary microbiome and CVD risk on a large cohort remain sparse. This study aims to identify whether a predictive salivary microbiome signature is associated with a high risk of developing CVD in the Qatari population. Methods: Saliva samples from 2,974 Qatar Genome Project (QGP) participants were collected from Qatar Biobank (QBB). Based on the CVD score, subjects were classified into low-risk (LR < 10) (n = 2491), moderate-risk (MR = 10-20) (n = 320) and high-risk (HR > 30) (n = 163). To assess the salivary microbiome (SM) composition, 16S-rDNA libraries were sequenced and analyzed using QIIME-pipeline. Machine Learning (ML) strategies were used to identify SM-based predictors of CVD risk. Results: Firmicutes and Bacteroidetes were the predominant phyla among all the subjects included. Linear Discriminant Analysis Effect Size (LEfSe) analysis revealed that Clostridiaceae and Capnocytophaga were the most significantly abundant genera in the LR group, while Lactobacillus and Rothia were significantly abundant in the HR group. ML based prediction models revealed that Desulfobulbus, Prevotella, and Tissierellaceae were the common predictors of increased risk to CVD. Conclusion: This study identified significant differences in the SM composition in HR and LR CVD subjects. This is the first study to apply ML-based prediction modeling using the SM to predict CVD in an Arab population. More studies are required to better understand the mechanisms of how those microbes contribute to CVD.

Bifidobacterium reduction is associated with high blood pressure in children with type 1 diabetes mellitus.

Children with Type 1 diabetes mellitus (T1DM) have an elevated risk of abnormal blood pressure (BP) measurements and patterns. Both hypertension and T1DM are well-known risk factors for cardiovascular disease and kidney failure. The human microbiome has been linked to both diabetes and hypertension, but the relationship between the gut microbiome and BP in children with T1DM is not well-understood. In this cross-sectional study, we examined the relationship between resting office BP and gut microbiota composition, diversity, and richness in children with T1DM and healthy controls. We recruited 29 pediatric subjects and divided them into three groups: healthy controls (HC, n = 5), T1DM with normal BP (T1DM-Normo, n = 17), and T1DM with elevated BP (T1DM-HBP, n = 7). We measured the BP, dietary and clinical parameters for each subject. We collected fecal samples to perform the 16s rDNA sequencing and to measure the short-chain fatty acids (SCFAs) level. The microbiome downstream analysis included the relative abundance of microbiota, alpha and beta diversity, microbial markers using Linear Discriminant effect size analysis (LEfSe), potential gut microbial metabolic pathways using Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) and metabolic pathways validation using Statistical Inference of Associations between Microbial Communities And host phenotype (SIAMCAT) machine learning toolbox. Our study results showed that T1DM-HBP group had distinct gut microbial composition (at multiple taxonomic levels) and reduced diversity (richness and abundance) compared with T1DM-Normo and HC groups. Children with T1DM-HBP showed a significant reduction of Bifidobacterium levels (especially B. adolescentis, B. bifidum, and B. longum) compared to the T1DM-Normo group. We also observed unique gut-microbial metabolic pathways, such as elevated lipopolysaccharide synthesis and glutathione metabolism in children with T1DM-HBP compared to T1DM-Normo children. We can conclude that the reduction in the abundance of genus Bifidobacterium could play a significant role in elevating the BP in pediatric T1DM subjects. More studies are needed to corroborate our findings and further explore the potential contributing mechanisms we describe.

Nutri-epigenetics: the effect of maternal diet and early nutrition on the pathogenesis of autoimmune diseases.

Autoimmune diseases comprise a wide group of diseases involving a self-response of the immune system against the host. The etiopathogenesis is very complex involving disease-specific factors but also environmental factors, among which the diet. Maternal diet during pregnancy as well as early nutrition recently attracted the interest of the scientists as contributing to the immune programming. In this paper, we reviewed the most recent literature on the effect of maternal diet and early nutrition in modulating the immune system in a selected subset of autoimmune diseases: type 1 diabetes, celiac disease, inflammatory bowel disease, juvenile idiopathic arthritis and rheumatoid arthritis. Particularly, we focused our narrative on the role of maternal and perinatal nutrition in the epigenetic mechanisms underlying the auto-immune response. Maternal diet during pregnancy as well as breastfeeding and early nutrition play a big role in many epigenetic mechanisms. Most of the nutrients consumed by the mother and the infant are known exerting epigenetic functions, such as folate, methionine, zinc, vitamins B12 and D, fibers, casein and gliadin, and they were linked to gene expression changes in the immune pathways. Despite the common role of maternal diet, breastfeeding and early nutrition in almost all the autoimmune diseases, each disease seems to have specific diet-driver epigenetic mechanisms that require further investigations. The research in this field is opening new routes to establishing a precision nutrition approach to the auto-immune diseases.

Akkermansia, a Possible Microbial Marker for Poor Glycemic Control in Qataris Children Consuming Arabic Diet-A Pilot Study on Pediatric T1DM in Qatar.

In Qatar, Type 1 Diabetes mellitus (T1DM) is one of the most prevalent disorders. This study aimed to explore the gut microbiome's relation to the continuous subcutaneous insulin infusion (CSII) therapy, dietary habits, and the HbA1c level in the pediatric T1DM subjects in Qatar. We recruited 28 T1DM subjects with an average age of 10.5 ± 3.53 years. The stool sample was used to measure microbial composition by 16s rDNA sequencing method. The results have revealed that the subjects who had undergone CSII therapy had increased microbial diversity and genus Akkermansia was significantly enriched in the subjects without CSII therapy. Moreover, genus Akkermansia was higher in the subjects with poor glycemic control (HbA1c > 7.5%). When we classified the subjects based on dietary patterns and nationality, Akkermansia was significantly enriched in Qataris subjects without the CSII therapy consuming Arabic diet than expatriates living in Qatar and eating a Western/mixed diet. Thus, this pilot study showed that abundance of Akkermansia is dependent on the Arabic diet only in poorly controlled Qataris T1DM patients, opening new routes to personalized treatment for T1DM in Qataris pediatric subjects. Further comprehensive studies on the relation between the Arabic diet, ethnicity, and Akkermansia are warranted to confirm this preliminary finding.

Annexin A3 in sepsis: novel perspectives from an exploration of public transcriptome data.

According to publicly available transcriptome datasets, the abundance of Annexin A3 (ANXA3) is robustly increased during the course of sepsis; however, no studies have examined the biological significance or clinical relevance of ANXA3 in this pathology. Here we explored this interpretation gap and identified possible directions for future research. Based on reference transcriptome datasets, we found that ANXA3 expression is restricted to neutrophils, is upregulated in vitro after exposure to plasma obtained from septic patients, and is associated with adverse clinical outcomes. Secondly, an increase in ANXA3 transcript abundance was also observed in vivo, in the blood of septic patients in multiple independent studies. ANXA3 is known to mediate calcium-dependent granules-phagosome fusion in support of microbicidal activity in neutrophils. More recent work has also shown that ANXA3 enhances proliferation and survival of tumour cells via a Caspase-3-dependent mechanism. And this same molecule is also known to play a critical role in regulation of apoptotic events in neutrophils. Thus, we posit that during sepsis ANXA3 might either play a beneficial role, by facilitating microbial clearance and resolution of the infection; or a detrimental role, by prolonging neutrophil survival, which is known to contribute to sepsis-mediated organ damage.

Comparison of Small Gut and Whole Gut Microbiota of First-Degree Relatives With Adult Celiac Disease Patients and Controls.

Recent studies on celiac disease (CeD) have reported alterations in the gut microbiome. Whether this alteration in the microbial community is the cause or effect of the disease is not well understood, especially in adult onset of disease. The first-degree relatives (FDRs) of CeD patients may provide an opportunity to study gut microbiome in pre-disease state as FDRs are genetically susceptible to CeD. By using 16S rRNA gene sequencing, we observed that ecosystem level diversity measures were not significantly different between the disease condition (CeD), pre-disease (FDR) and control subjects. However, differences were observed at the level of amplicon sequence variant (ASV), suggesting alterations in specific ASVs between pre-disease and diseased condition. Duodenal biopsies showed higher differences in ASVs compared to fecal samples indicating larger disruption of the microbiota at the disease site. The duodenal microbiota of FDR was characterized by significant abundance of ASVs belonging to Parvimonas, Granulicatella, Gemella, Bifidobacterium, Anaerostipes, and Actinomyces genera. The duodenal microbiota of CeD was characterized by higher abundance of ASVs from genera Megasphaera and Helicobacter compared to the FDR microbiota. The CeD and FDR fecal microbiota had reduced abundance of ASVs classified as Akkermansia and Dorea when compared to control group microbiota. In addition, predicted functional metagenome showed reduced ability of gluten degradation by CeD fecal microbiota in comparison to FDRs and controls. The findings of the present study demonstrate differences in ASVs and predicts reduced ability of CeD fecal microbiota to degrade gluten compared to the FDR fecal microbiota. Further research is required to investigate the strain level and active functional profiles of FDR and CeD microbiota to better understand the role of gut microbiome in pathophysiology of CeD.

Identification of CETP as a molecular target for estrogen positive breast cancer cell death by cholesterol depleting agents.

Cholesterol and its metabolites act as steroid hormone precursors, which promote estrogen receptor positive (ER+) breast cancer (BC) progression. Development of cholesterol targeting anticancer drugs has been hindered due to the lack of knowledge of viable molecular targets. Till now, Cholesteryl ester transfer protein (CETP) has been envisaged as a feasible molecular target in atherosclerosis, but for the first time, we show that CETP contributes to BC cell survival when challenged with cholesterol depleting agents. We show that MCF-7 CETP knockout BC cells pose less resistance towards cytotoxic compounds (Tamoxifen and Acetyl Plumbagin (AP)), and were more susceptible to intrinsic apoptosis. Analysis of differentially expressed genes using Ingenuity Pathway Analysis (IPA), in vivo tumor inhibition, and in vitro phenotypic responses to AP revealed a unique CETP-centric cholesterol pathway involved in sensitizing ER+ BC cells to intrinsic mitochondrial apoptosis. Furthermore, analysis of cell line, tissue and patient data available in publicly available databases linked elevated CETP expression to cancer, cancer relapse and overall poor survival. Overall, our findings highlight CETP as a pharmacologically relevant and unexploited cellular target in BC. The work also highlights AP as a promising chemical entity for preclinical investigations as a cholesterol depleting anticancer therapeutic agent.

The Arabidopsis RNA-binding protein AtRGGA regulates tolerance to salt and drought stress.

Salt and drought stress severely reduce plant growth and crop productivity worldwide. The identification of genes underlying stress response and tolerance is the subject of intense research in plant biology. Through microarray analyses, we previously identified in potato (Solanum tuberosum) StRGGA, coding for an Arginine Glycine Glycine (RGG) box-containing RNA-binding protein, whose expression was specifically induced in potato cell cultures gradually exposed to osmotic stress. Here, we show that the Arabidopsis (Arabidopsis thaliana) ortholog, AtRGGA, is a functional RNA-binding protein required for a proper response to osmotic stress. AtRGGA gene expression was up-regulated in seedlings after long-term exposure to abscisic acid (ABA) and polyethylene glycol, while treatments with NaCl resulted in AtRGGA down-regulation. AtRGGA promoter analysis showed activity in several tissues, including stomata, the organs controlling transpiration. Fusion of AtRGGA with yellow fluorescent protein indicated that AtRGGA is localized in the cytoplasm and the cytoplasmic perinuclear region. In addition, the rgga knockout mutant was hypersensitive to ABA in root growth and survival tests and to salt stress during germination and at the vegetative stage. AtRGGA-overexpressing plants showed higher tolerance to ABA and salt stress on plates and in soil, accumulating lower levels of proline when exposed to drought stress. Finally, a global analysis of gene expression revealed extensive alterations in the transcriptome under salt stress, including several genes such as ASCORBATE PEROXIDASE2, GLUTATHIONE S-TRANSFERASE TAU9, and several SMALL AUXIN UPREGULATED RNA-like genes showing opposite expression behavior in transgenic and knockout plants. Taken together, our results reveal an important role of AtRGGA in the mechanisms of plant response and adaptation to stress.

Activities and specificities of homodimeric TALENs in Saccharomyces cerevisiae.

The development of highly efficient genome engineering reagents is of paramount importance to launch the next wave of biotechnology. TAL effectors have been developed as an adaptable DNA binding scaffold that can be engineered to bind to any user-defined sequence. Thus, TAL-based DNA binding modules have been used to generate chimeric proteins for a variety of targeted genome modifications across eukaryotic species. For example, TAL effectors fused to the catalytic domain of FokI endonuclease (TALENs) were used to generate site-specific double strand breaks (DSBs), the repair of which can be harnessed to dictate user-desired, genome-editing outcomes. To cleave DNA, FokI endonuclease must dimerize which can be achieved using a pair of TALENs that bind to the DNA targeted in a tail-to-tail orientation with proper spacing allowing the dimer formation. Because TALENs binding to DNA are dependent on their repeat sequences and nucleotides binding specificities, homodimers and heterodimers binding can be formed. In the present study, we used several TALEN monomers with increased repeats binding degeneracy to allow homodimer formation at increased number of genomic loci. We assessed their binding specificities and genome modification activities. Our results indicate that homodimeric TALENs could be used to modify the yeast genome in a site-specific manner and their binding to the promoter regions might modulate the expression of target genes. Taken together, our data indicate that homodimeric TALENs could be used to achieve different engineering possibilities of biotechnological applications and that their transcriptional modulations need to be considered when analyzing their phenotypic effects.

Arabidopsis ECERIFERUM9 involvement in cuticle formation and maintenance of plant water status.

Mutation of the ECERIFERUM9 (CER9) gene in Arabidopsis (Arabidopsis thaliana) causes elevated amounts of 18-carbon-length cutin monomers and a dramatic shift in the cuticular wax profile (especially on leaves) toward the very-long-chain free fatty acids tetracosanoic acid (C24) and hexacosanoic acid (C26). Relative to the wild type, cer9 mutants exhibit elevated cuticle membrane thickness over epidermal cells and cuticular ledges with increased occlusion of the stomatal pore. The cuticular phenotypes of cer9 are associated with delayed onset of wilting in plants experiencing water deficit, lower transpiration rates, and improved water use efficiency measured as carbon isotope discrimination. The CER9 protein thus encodes a novel determinant of plant drought tolerance-associated traits, one whose deficiency elevates cutin synthesis, redistributes wax composition, and suppresses transpiration. Map-based cloning identified CER9, and sequence analysis predicted that it encodes an E3 ubiquitin ligase homologous to yeast Doa10 (previously shown to target endoplasmic reticulum proteins for proteasomal degradation). To further elucidate CER9 function, the impact of CER9 deficiency on interactions with other genes was examined using double mutant and transcriptome analyses. For both wax and cutin, cer9 showed mostly additive effects with cer6, long-chain acyl-CoA synthetase1 (lacs1), and lacs2 and revealed its role in early steps of both wax and cutin synthetic pathways. Transcriptome analysis revealed that the cer9 mutation affected diverse cellular processes, with primary impact on genes associated with diverse stress responses. The discovery of CER9 lays new groundwork for developing novel cuticle-based strategies for improving the drought tolerance and water use efficiency of crop plants.

Targeted transcriptional repression using a chimeric TALE-SRDX repressor protein.

Transcriptional activator-like effectors (TALEs) are proteins secreted by Xanthomonas bacteria when they infect plants. TALEs contain a modular DNA binding domain that can be easily engineered to bind any sequence of interest, and have been used to provide user-selected DNA-binding modules to generate chimeric nucleases and transcriptional activators in mammalian cells and plants. Here we report the use of TALEs to generate chimeric sequence-specific transcriptional repressors. The dHax3 TALE was used as a scaffold to provide a DNA-binding module fused to the EAR-repression domain (SRDX) to generate a chimeric repressor that targets the RD29A promoter. The dHax3.SRDX protein efficiently repressed the transcription of the RD29A::LUC transgene and endogenous RD29A gene in Arabidopsis. Genome wide expression profiling showed that the chimeric repressor also inhibited the expression of several other genes that contain the designer TALE-target sequence in their promoters. Our data suggest that TALEs can be used to generate chimeric repressors to specifically repress the transcription of genes of interest in plants. This sequence-specific transcriptional repression by direct on promoter effector technology is a powerful tool for functional genomics studies and biotechnological applications.

The glossyhead1 allele of ACC1 reveals a principal role for multidomain acetyl-coenzyme A carboxylase in the biosynthesis of cuticular waxes by Arabidopsis.

A novel mutant of Arabidopsis (Arabidopsis thaliana), having highly glossy inflorescence stems, postgenital fusion in floral organs, and reduced fertility, was isolated from an ethyl methanesulfonate-mutagenized population and designated glossyhead1 (gsd1). The gsd1 locus was mapped to chromosome 1, and the causal gene was identified as a new allele of Acetyl-Coenzyme A Carboxylase1 (ACC1), a gene encoding the main enzyme in cytosolic malonyl-coenzyme A synthesis. This, to our knowledge, is the first mutant allele of ACC1 that does not cause lethality at the seed or early germination stage, allowing for the first time a detailed analysis of ACC1 function in mature tissues. Broad lipid profiling of mature gsd1 organs revealed a primary role for ACC1 in the biosynthesis of the very-long-chain fatty acids (C(20:0) or longer) associated with cuticular waxes and triacylglycerols. Unexpectedly, transcriptome analysis revealed that gsd1 has limited impact on any lipid metabolic networks but instead has a large effect on environmental stress-responsive pathways, especially senescence and ethylene synthesis determinants, indicating a possible role for the cytosolic malonyl-coenzyme A-derived lipids in stress response signaling.

Prediction of clinical outcome in multiple lung cancer cohorts by integrative genomics: implications for chemotherapy selection.

The role of adjuvant chemotherapy in patients with stage IB non-small-cell lung cancer (NSCLC) is controversial. Identifying patient subgroups with the greatest risk of relapse and, consequently, most likely to benefit from adjuvant treatment thus remains an important clinical challenge. Here, we hypothesized that recurrent patterns of genomic amplifications and deletions in lung tumors could be integrated with gene expression information to establish a robust predictor of clinical outcome in stage IB NSCLC. Using high-resolution microarrays, we generated tandem DNA copy number and gene expression profiles for 85 stage IB lung adenocarcinomas/large cell carcinomas. We identified specific copy number alterations linked to relapse-free survival and selected genes within these regions exhibiting copy number-driven expression to construct a novel integrated signature (IS) capable of predicting clinical outcome in this series (P = 0.02). Importantly, the IS also significantly predicted clinical outcome in two other independent stage I NSCLC cohorts (P = 0.003 and P = 0.025), showing its robustness. In contrast, a more conventional molecular predictor based solely on gene expression, while capable of predicting outcome in the initial series, failed to significantly predict outcome in the two independent data sets. Our results suggest that recurrent copy number alterations, when combined with gene expression information, can be successfully used to create robust predictors of clinical outcome in early-stage NSCLC. The utility of the IS in identifying early-stage NSCLC patients as candidates for adjuvant treatment should be further evaluated in a clinical trial.

Functional analysis of a cell cycle-associated, tumor-suppressive gene, protein tyrosine phosphatase receptor type G, in nasopharyngeal carcinoma.

Functional studies to identify the potential role of a chromosome 3p14-21 gene, protein tyrosine phosphatase receptor type G (PTPRG), were performed. PTPRG was identified as a candidate tumor suppressor gene (TSG) in nasopharyngeal carcinoma (NPC) by differential gene profiling of tumorigenic and nontumorigenic NPC chromosome 3 microcell hybrids (MCH). Down-regulation of this gene was found in tumor segregants when compared with their corresponding tumor-suppressive MCHs, as well as in NPC cell lines and tumor biopsies. Promoter hypermethylation and loss of heterozygosity were found to be important mechanisms contributing to PTPRG silencing. PTPRG overexpression in NPC cell lines induces growth suppression and reduced anchorage-independent growth in vitro. This is the first study to use a tetracycline-responsive vector expression system to study PTPRG stable transfectants. Results indicate its ability to induce significant tumor growth suppression in nude mice under conditions activating transgene expression. These studies now provide functional evidence indicating critical interactions of PTPRG in the extracellular matrix milieu induce cell arrest and changes in cell cycle status. This is associated with inhibition of pRB phosphorylation through down-regulation of cyclin D1. These novel findings enhance our current understanding of how PTPRG may contribute to tumorigenesis.

Human and mouse oligonucleotide-based array CGH.

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using 'off-the-shelf' oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.

THY1 is a candidate tumour suppressor gene with decreased expression in metastatic nasopharyngeal carcinoma.

Using oligonucleotide microarray analysis, THY1, mapping close to a previously defined 11q22-23 nasopharyngeal carcinoma (NPC) critical region was identified as showing consistent downregulated expression in the tumour segregants, as compared to their parental tumour-suppressing microcell hybrids (MCHs). Gene expression and protein analyses show that THY1 was not expressed in the NPC HONE1 recipient cells, tumour segregants, and other NPC cell lines; THY1 was exclusively expressed in the non-tumourigenic MCHs. The mechanism of THY1 gene inactivation in these cell lines was attributed to hypermethylation. Clinical study showed that in 65% of NPC specimens there was either downregulation or loss of THY1 gene expression. Using a tissue microarray and immunohistochemical staining, 44% of the NPC cases showed downregulated expression of THY1 and 9% lost THY1 expression. The frequency of THY1 downregulated expression in lymph node metastatic NPC was 63%, which was significantly higher than in the primary tumour (33%). After transfection of THY1 gene into HONE1 cells, a dramatic reduction of colony formation ability was observed. These findings suggest that THY1 is a good candidate tumour suppressor gene in NPC, which is significantly associated with lymph node metastases.

Discovery of estrogen receptor alpha target genes and response elements in breast tumor cells.

BACKGROUND: Estrogens and their receptors are important in human development, physiology and disease. In this study, we utilized an integrated genome-wide molecular and computational approach to characterize the interaction between the activated estrogen receptor (ER) and the regulatory elements of candidate target genes. RESULTS: Of around 19,000 genes surveyed in this study, we observed 137 ER-regulated genes in T-47D cells, of which only 89 were direct target genes. Meta-analysis of heterogeneous in vitro and in vivo datasets showed that the expression profiles in T-47D and MCF-7 cells are remarkably similar and overlap with genes differentially expressed between ER-positive and ER-negative tumors. Computational analysis revealed a significant enrichment of putative estrogen response elements (EREs) in the cis-regulatory regions of direct target genes. Chromatin immunoprecipitation confirmed ligand-dependent ER binding at the computationally predicted EREs in our highest ranked ER direct target genes, NRIP1, GREB1 and ABCA3. Wider examination of the cis-regulatory regions flanking the transcriptional start sites showed species conservation in mouse-human comparisons in only 6% of predicted EREs. CONCLUSIONS: Only a small core set of human genes, validated across experimental systems and closely associated with ER status in breast tumors, appear to be sufficient to induce ER effects in breast cancer cells. That cis-regulatory regions of these core ER target genes are poorly conserved suggests that different evolutionary mechanisms are operative at transcriptional control elements than at coding regions. These results predict that certain biological effects of estrogen signaling will differ between mouse and human to a larger extent than previously thought.