Ultraconserved Regions Research Articles

Integration of major histocompatibility complex, methylation, and transcribed ultra-conserved regions analyses in uremia

Integration of major histocompatibility complex, methylation, and transcribed ultra-conserved regions analyses in uremia

The role of non-coding, transcribed ultra-conserved regions as tissue biomarkers in cancers

The role of non-coding, transcribed ultra-conserved regions as tissue biomarkers in cancers

NcPred: ncRNA-Disease Association Prediction through Tripartite Network-Based Inference.

Motivation: Over the past few years, experimental evidence has highlighted the role of microRNAs to human diseases. miRNAs are critical for the regulation of cellular processes, and, therefore, their aberration can be among the triggering causes of pathological phenomena. They are just one member of the large class of non-coding RNAs, which include transcribed ultra-conserved regions (T-UCRs), small nucleolar RNAs (snoRNAs), PIWI-interacting RNAs (piRNAs), large intergenic non-coding RNAs (lincRNAs) and, the heterogeneous group of long non-coding RNAs (lncRNAs). Their associations with diseases are few in number, and their reliability is questionable. In literature, there is only one recent method proposed by Yang et al. (2014) to predict lncRNA-disease associations. This technique, however, lacks in prediction quality. All these elements entail the need to investigate new bioinformatics tools for the prediction of high quality ncRNA-disease associations. Here, we propose a method called ncPred for the inference of novel ncRNA-disease association based on recommendation technique. We represent our knowledge through a tripartite network, whose nodes are ncRNAs, targets, or diseases. Interactions in such a network associate each ncRNA with a disease through its targets. Our algorithm, starting from such a network, computes weights between each ncRNA-disease pair using a multi-level resource transfer technique that at each step takes into account the resource transferred in the previous one.Results: The results of our experimental analysis show that our approach is able to predict more biologically significant associations with respect to those obtained by Yang et al. (2014), yielding an improvement in terms of the average area under the ROC curve (AUC). These results prove the ability of our approach to predict biologically significant associations, which could lead to a better understanding of the molecular processes involved in complex diseases.Availability: All the ncPred predictions together with the datasets used for the analysis are available at the following url: http://alpha.dmi.unict.it/ncPred/

A large scale expression study associates uc.283-plus lncRNA with pluripotent stem cells and human glioma.

BackgroundThere are 481 ultra-conserved regions (UCRs) longer than 200 bases in the genomes of human, mouse and rat. These DNA sequences are absolutely conserved and show 100% identity with no insertions or deletions. About half of these UCRs are reported as transcribed and many correspond to long non-coding RNAs (lncRNAs).MethodsWe used custom microarrays with 962 probes representing sense and antisense sequences for the 481 UCRs to examine their expression across 374 normal samples from 46 different tissues and 510 samples representing 10 different types of cancer. The expression in embryonic stem cells of selected UCRs was validated by real time PCR.ResultsWe identified tissue selective UCRs and studied UCRs in embryonic and induced pluripotent stem cells. Among the normal tissues, the uc.283 lncRNA was highly specific for pluripotent stem cells. Intriguingly, the uc.283-plus lncRNA was highly expressed in some solid cancers, particularly in one of the most untreatable types, glioma.ConclusionOur results suggest that uc.283-plus lncRNA might have a role in pluripotency of stem cells and in the biology of glioma.Electronic supplementary materialThe online version of this article (doi:10.1186/s13073-014-0076-4) contains supplementary material, which is available to authorized users.

Transcribed ultraconserved noncoding RNAs (T-UCR) are involved in Barrett's esophagus carcinogenesis.

Barrett's esophagus (BE) involves a metaplastic replacement of native esophageal squamous epithelium (Sq) by columnar-intestinalized mucosa, and it is the main risk factor for Barrett-related adenocarcinoma (BAc). Ultra-conserved regions (UCRs) are a class non-coding sequences that are conserved in humans, mice and rats. More than 90% of UCRs are transcribed (T-UCRs) in normal tissues, and are altered at transcriptional level in tumorigenesis. To identify the T-UCR profiles that are dysregulated in Barrett's mucosa transformation, microarray analysis was performed on a discovery set of 51 macro-dissected samples obtained from 14 long-segment BE patients. Results were validated in an independent series of esophageal biopsy/surgery specimens and in two murine models of Barrett's esophagus (i.e. esophagogastric-duodenal anastomosis). Progression from normal to BE to adenocarcinoma was each associated with specific and mutually exclusive T-UCR signatures that included up-regulation of uc.58-, uc.202-, uc.207-, and uc.223- and down-regulation of uc.214+. A 9 T-UCR signature characterized BE versus Sq (with the down-regulation of uc.161-, uc.165-, and uc.327-, and the up-regulation of uc.153-, uc.158-, uc.206-, uc.274-, uc.472-, and uc.473-). Analogous BE-specific T-UCR profiles were shared by human and murine lesions. This study is the first demonstration of a role for T-UCRs in the transformation of Barrett's mucosa.

An Ultraconserved lnc to miRNA Processing

Very few specific functions have been assigned to ultraconserved regions. In this issue of Molecular Cell, Liz et al. (2014) describe how a lncRNA transcribed from an ultraconserved region can negatively regulate miRNA maturation.

Expression of transcribed ultraconserved regions of genome in rat cerebral cortex

Emerging evidence indicates that 481 regions of the genome (>200bp) that actively transcribe noncoding RNAs shows 100% homology between humans, rats and mice. These transcribed ultraconserved regions (T-UCRs) are thought to control the essential regulatory functions basic for life in rodents and mammals. Using microarray analysis, we presently show that 107 T-UCRs are actively expressed in adult rat cerebral cortex. They are grouped into intragenic (61) and intergenic (46) based on their genic location. Interestingly, 10 T-UCRs are expressed at unusually high levels in cerebral cortex. Additionally, many T-UCRs also showed cogenic expression. We further analyzed the correlation of intragenic T-UCRs with their host protein coding genes. Surprisingly, most of the expressed intragenic T-UCRs (54 out of 61) displayed a negative correlation with their host gene expression. T-UCRs are thought to control the splicing and transcription of the protein-coding genes that host them and flank them. Bioinformatics analysis indicated that the protein products of majority of these genes are nuclear in localization, share protein domains and are involved in the regulation of diverse biological and molecular functions including metabolism, development, cell cycle, binding and transcription factor regulation. In conclusion, this is the first study to shows that many T-UCRs are expressed in rodent brain and they might play a role in physiological brain functions.

A large scale expression study associates uc.283-plus lncRNA with pluripotent stem cells and human glioma

BackgroundThere are 481 ultra-conserved regions (UCRs) longer than 200 bases in the genomes of human, mouse and rat. These DNA sequences are absolutely conserved and show 100% identity with no insertions or deletions. About half of these UCRs are reported as transcribed and many correspond to long non-coding RNAs (lncRNAs).MethodsWe used custom microarrays with 962 probes representing sense and antisense sequences for the 481 UCRs to examine their expression across 374 normal samples from 46 different tissues and 510 samples representing 10 different types of cancer. The expression in embryonic stem cells of selected UCRs was validated by real time PCR.ResultsWe identified tissue selective UCRs and studied UCRs in embryonic and induced pluripotent stem cells. Among the normal tissues, the uc.283 lncRNA was highly specific for pluripotent stem cells. Intriguingly, the uc.283-plus lncRNA was highly expressed in some solid cancers, particularly in one of the most untreatable types, glioma.ConclusionOur results suggest that uc.283-plus lncRNA might have a role in pluripotency of stem cells and in the biology of glioma.

A tumor-promoting mechanism mediated by retrotransposon-encoded reverse transcriptase is active in human transformed cell lines

LINE-1 elements make up the most abundant retrotransposon family in the human genome. Full-length LINE-1 elements encode a reverse transcriptase (RT) activity required for their own retrotranpsosition as well as that of non-autonomous Alu elements. LINE-1 are poorly expressed in normal cells and abundantly in cancer cells. Decreasing RT activity in cancer cells, by either LINE-1-specific RNA interference, or by RT inhibitory drugs, was previously found to reduce proliferation and promote differentiation and to antagonize tumor growth in animal models. Here we have investigated how RT exerts these global regulatory functions. We report that the RT inhibitor efavirenz (EFV) selectively downregulates proliferation of transformed cell lines, while exerting only mild effects on non-transformed cells; this differential sensitivity matches a differential RT abundance, which is high in the former and undetectable in the latter. Using CsCl density gradients, we selectively identify Alu and LINE-1 containing DNA:RNA hybrid molecules in cancer but not in normal cells. Remarkably, hybrid molecules fail to form in tumor cells treated with EFV under the same conditions that repress proliferation and induce the reprogramming of expression profiles of coding genes, microRNAs (miRNAs) and ultraconserved regions (UCRs). The RT-sensitive miRNAs and UCRs are significantly associated with Alu sequences. The results suggest that LINE-1-encoded RT governs the balance between single-stranded and double-stranded RNA production. In cancer cells the abundant RT reverse-transcribes retroelement-derived mRNAs forming RNA:DNA hybrids. We propose that this impairs the formation of double-stranded RNAs and the ensuing production of small regulatory RNAs, with a direct impact on gene expression. RT inhibition restores the 'normal' small RNA profile and the regulatory networks that depend on them. Thus, the retrotransposon-encoded RT drives a previously unrecognized mechanism crucial to the transformed state in tumor cells.

HINCUTs in cancer: hypoxia-induced noncoding ultraconserved transcripts

Recent data have linked hypoxia, a classic feature of the tumor microenvironment, to the function of specific microRNAs (miRNAs); however, whether hypoxia affects other types of noncoding transcripts is currently unknown. Starting from a genome-wide expression profiling, we demonstrate for the first time a functional link between oxygen deprivation and the modulation of long noncoding transcripts from ultraconserved regions, termed transcribed-ultraconserved regions (T-UCRs). Interestingly, several hypoxia-upregulated T-UCRs, henceforth named 'hypoxia-induced noncoding ultraconserved transcripts' (HINCUTs), are also overexpressed in clinical samples from colon cancer patients. We show that these T-UCRs are predominantly nuclear and that the hypoxia-inducible factor (HIF) is at least partly responsible for the induction of several members of this group. One specific HINCUT, uc.475 (or HINCUT-1) is part of a retained intron of the host protein-coding gene, O-linked N-acetylglucosamine transferase, which is overexpressed in epithelial cancer types. Consistent with the hypothesis that T-UCRs have important function in tumor formation, HINCUT-1 supports cell proliferation specifically under hypoxic conditions and may be critical for optimal O-GlcNAcylation of proteins when oxygen tension is limiting. Our data gives a first glimpse of a novel functional hypoxic network comprising protein-coding transcripts and noncoding RNAs (ncRNAs) from the T-UCRs category.

Transcription signatures encoded by ultraconserved genomic regions in human prostate cancer

BackgroundUltraconserved regions (UCR) are genomic segments of more than 200 base pairs that are evolutionarily conserved among mammalian species. They are thought to have functions as transcriptional enhancers and regulators of alternative splicing. Recently, it was shown that numerous RNAs are transcribed from these regions. These UCR-encoded transcripts (ucRNAs) were found to be expressed in a tissue- and disease-specific manner and may interfere with the function of other RNAs through RNA: RNA interactions. We hypothesized that ucRNAs have unidentified roles in the pathogenesis of human prostate cancer. In a pilot study, we examined ucRNA expression profiles in human prostate tumors.MethodsUsing a custom microarray with 962 probesets representing sense and antisense sequences for the 481 human UCRs, we examined ucRNA expression in resected, fresh-frozen human prostate tissues (57 tumors, 7 non-cancerous prostate tissues) and in cultured prostate cancer cells treated with either epigenetic drugs (the hypomethylating agent, 5-Aza 2′deoxycytidine, and the histone deacetylase inhibitor, trichostatin A) or a synthetic androgen, R1881. Expression of selected ucRNAs was also assessed by qRT-PCR and NanoString®-based assays. Because ucRNAs may function as RNAs that target protein-coding genes through direct and inhibitory RNA: RNA interactions, computational analyses were applied to identify candidate ucRNA:mRNA binding pairs.ResultsWe observed altered ucRNA expression in prostate cancer (e.g., uc.106+, uc.477+, uc.363 + A, uc.454 + A) and found that these ucRNAs were associated with cancer development, Gleason score, and extraprostatic extension after controlling for false discovery (false discovery rate < 5% for many of the transcripts). We also identified several ucRNAs that were responsive to treatment with either epigenetic drugs or androgen (R1881). For example, experiments with LNCaP human prostate cancer cells showed that uc.287+ is induced by R1881 (P < 0.05) whereas uc.283 + A was up-regulated following treatment with combined 5-Aza 2′deoxycytidine and trichostatin A (P < 0.05). Additional computational analyses predicted RNA loop-loop interactions of 302 different sense and antisense ucRNAs with 1058 different mRNAs, inferring possible functions of ucRNAs via direct interactions with mRNAs.ConclusionsThis first study of ucRNA expression in human prostate cancer indicates an altered transcript expression in the disease.

Transcribed ultraconserved regions are aberrantly expressed and can be modulated by interleukin 6 in cholangiocarcinoma

BackgroundThe inflammation-associated cytokine interleukin (IL) 6 has been implicated in cholangiocarcinogenesis. Transcripts encoded by a group of highly conserved regions (transcribed-ultraconserved regions [T-UCRs]) are novel promising non-coding RNAs that proved to have a role in human carcinogenesis and liver cancer progression. We aimed to study expression of T-UCRs in cholangiocarcinoma. MethodsMzChA-1, TFK, CC-LP-1, and KMCH human cholangiocarcinoma cells, and normal human intrahepatic biliary epithelial cells (HIBEC) were used. T-UCR expression profiling was done with real-time PCR. IL-6 dependent T-UCR expression was assessed in Mz-ChA-1 cells incubated with 10 ng/L IL6 for 24 h or stably transfected with full-length IL6 (Mz-IL6). FindingsThe expression of 65 T-UCRs was altered in Mz-ChA-1 compared with normal HIBEC cells. Of these, 12 T-UCRs were upregulated by more than two fold and 48 T-UCRs were downregulated including ultraconserved RNA uc.169, which was dramatically reduced in other human cholangiocarcinoma cell lines and tissues. uc.169 was further reduced in Mz-ChA-1 cells overexpressing IL6. Incubation of MzChA-1 cells with IL6 reduced expression of uc.169, and pretreatment with the inhibitor of transcription actinomycin D inhibited this effect. Enforced expression of IL6 caused an increase in the microRNAs miR-21 and miR-26-a1, which are involved in promotion of cholangiocarcinoma. The uc.169 sequence includes target seed regions for miR-21 and miR-26-a1 and they may be responsible for the downregulation of uc.169. Overexpression of uc.169 in Mz-ChA-1 cells reduced cell proliferation and caused arrest in phase G0/1 of the cell cycle, with increased apoptosis. InterpretationWe report that T-UCRs are differentially expressed in malignant human cholangiocytes, and that IL6 can modulate the expression of selected T-UCRs, probably through a miRNA-mediated modulation. These studies highlight for the first time a role of ultraconserved non-coding RNAs as novel effectors of IL-6 signalling, and the involvement of these RNA genes in the pathogenesis of cholangiocarcinoma. They lay the bases to further investigation of the clinical implications of T-UCR in human cholangiocarcinoma. FundingUniversity of Glasgow.

CpG island hypermethylation-associated silencing of small nucleolar RNAs in human cancer

Much effort in cancer research has focused on the tiny part of our genome that codes for mRNA. However, it has recently been recognized that microRNAs also contribute decisively to tumorigenesis. Studies have also shown that epigenetic silencing by CpG island hypermethylation of microRNAs with tumor suppressor activities is a common feature of human cancer. The importance of other classes of non-coding RNAs, such as long intergenic ncRNAs (lincRNAs) and transcribed ultraconserved regions (T-UCRs) as altered elements in neoplasia, is also gaining recognition. Thus, we wondered whether there were other ncRNAs undergoing CpG island hypermethylation-associated inactivation in cancer cells. We focused on the small nucleolar RNAs (snoRNAs), a subset of ncRNA with a wide variety of cellular functions, such as chemical modification of RNA, pre-RNA processing and control of alternative splicing. By data mining snoRNA databases and the scientific literature, we selected 49 snoRNAs that had a CpG island within ≤ 2 Kb or that were processed from a host gene with a 5′-CpG island. Bisulfite genomic sequencing of multiple clones in normal colon mucosa and the colorectal cancer cell line HCT-116 showed that 46 snoRNAs were equally methylated in the two samples: completely unmethylated (n = 26) or fully methylated (n = 20). Most interestingly, the host gene-associated 5′-CpG islands of the snoRNAs SNORD123, U70C and ACA59B were hypermethylated in the cancer cells but not in the corresponding normal tissue. CpG island hypermethylation was associated with the transcriptional silencing of the respective snoRNAs. Results of a DNA methylation microarray platform in a comprehensive collection of normal tissues, cancer cell lines and primary malignancies demonstrated that the observed hypermethylation of snoRNAs was a common feature of various tumor types, particularly in leukemias. Overall, our findings indicate the existence of a new subclass of ncRNAs, snoRNAs, that are targeted by epigenetic inactivation in human cancer.

Abstract 202: Expressional alterations in functional ultra-conserved non-coding RNAs in response to ATRA-induced differentiation and changes in MYCN levels in neuroblastoma cells

Abstract Neuroblastoma (NB) is a childhood cancer arising from precursor cells of the sympathetic nervous system with highly heterogeneous clinical behaviour, ranging from spontaneous regression to rapid progression and death due to disease. Amplification and over-expression of the MYCN transcription factor in NB tumors is highly correlated with poor survival. Children with high risk NB are given the synthetic retinoic acid, 13-cis-retinoic acid to reduce minimal residual disease, and exposure of a number of NB cell lines to the compound all-trans-retinoic acid (ATRA) induces neural differentiation along with the down-regulation of MYCN. Thus, identification of targets regulated by either ATRA or MYCN is of potential interest for the development of novel therapeutics. Ultra-conserved regions (UCRs) are sequences ≥200bp in length that are 100% conserved between human, mouse and rat genomes. In NB, transcribed UCRs (T-UCRs) exhibit specific profiles in MYCN-amplified (MNA) compared to non-MNA tumors (Mestdagh et al., Oncogene 2010 29:3583), and in high risk versus low risk tumors (Scaruffi et al., BMC Cancer 2009 9;441). Here we identify T-UCRs that are responsive to ATRA as well as T-UCRs that are directly or indirectly regulated by MYCN, and evaluate the biological significance of selected T-UCRs in functional studies of NB cell lines. We designed a custom tiling microarray to profile the expression of 481 UCRs in sense and antisense orientation (962 potential T-UCRs) in three ATRA-sensitive NB cell lines, and in SHEP-21N cells which contain a MYCN trans-gene controlled by a tetracycline responsive repressor element. Thirty-two T-UCRs were differentially expressed following ATRA treatment across all three ATRA sensitive cell lines (p&amp;lt;0.05), while analysis of doxycyclin-treated SHEP-21N cells (to deplete MYCN levels) indicated that 42 T-UCRs were altered by changing MYCN levels. In addition we identified a small number of T-UCRs, including T-UC.396 (intergenic) and T-UC.324 (antisense to host gene), whose expression was altered in both systems. These T-UCRs may represent independent ncRNAs directly or indirectly regulated by MYCN. Functionality of T-UC.300A and T-UC.324 was assessed by siRNA knockdown. While knockdown of T-UC.324 had no effect on cell viability or invasion, depletion of T-UC.300A resulted in a significant decrease in both cell viability and invasion. Our results indicate that significant numbers of T-UCRs have altered expression levels in response to changing MYCN levels and ATRA-induced differentiation. While the precise roles that these ncRNAs might play in cancer or in normal development are largely unknown, we have identified a T-UCR, T-UC.300A, whose down-regulation results in the decreased viability and invasiveness of ATRA-responsive cell lines and may play a functional role in ATRA-induced differentiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 202. doi:1538-7445.AM2012-202

Noncoding RNAs: Different roles in tumorigenesis

A major portion of the mammalian genome is transcribed to produce large numbers of noncoding RNAs (ncRNAs). During the past decade, the discovery of small RNAs, including the microRNAs (miRNA) and small interfering RNAs (siRNA), has led to important advances in biology. The breadth of the ncRNA field of study has substantially expanded and many recent results have revealed a range of functions that can be attributed to the miRNAs and other ncRNAs. For example, H19 RNA, HOTAIR RNA, transcribed ultraconserved regions (T-UCRs), natural antisense RNA, transfer RNA and mitochondrial noncoding RNA have been suggested to play important roles in cancers and other diseases as well as in diverse cellular processes. In this review, we focus on the current status of several classes of ncRNAs associated with cancer with the emphasis on those that are not microRNAs.

Expression Levels of Transcribed Ultraconserved Regions uc.73 and uc.388 Are Altered in Colorectal Cancer

Objectives: The development of colorectal cancer (CRC) is characterized by multiple genetic alterations. Transcribed ultraconserved regions (T-UCRs) are a subset of 481 sequenc es longer than 200 bp, which are absolutely conserved between orthologous regions of human, rat and mouse genomes, and are actively transcribed. It has recently been proven in cancer systems that differentially expressed T-UCRs could alter the functional characteristics of malignant cells. Genome-wide profiling revealed that T-UCRs have distinct signatures in human leukemia and carcinoma. Methods: In our study, we examined the expression levels of uc.43, uc.73, uc.134, uc.230, uc.339, uc.388 and uc.399 in 54 samples of primary colorectal carcinomas and 15 samples of non-tumoral adjacent tissues by real-time PCR. T-UCR expression levels were also correlated with commonly used clinicopathological features of CRC. Results: Expression levels of uc.73 (p = 0.0139) and uc.388 (p = 0.0325) were significantly decreased in CRC tissue, and uc.73 indicated a positive correlation with overall survival (p = 0.0315). The lower expression of uc.388 was associated with the distal location of CRC (p = 0.0183), but no correlation of any evaluated T-UCR with clinical stage, grade and tumor diameter was observed. Conclusion: Our preliminary results suggest that uc.73 and uc.388 could be potential diagnostic and prognostic biomarkers in CRC patients.

Non-coding RNAs in human disease

The relevance of the non-coding genome to human disease has mainly been studied in the context of the widespread disruption of microRNA (miRNA) expression and function that is seen in human cancer. However, we are only beginning to understand the nature and extent of the involvement of non-coding RNAs (ncRNAs) in disease. Other ncRNAs, such as PIWI-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), transcribed ultraconserved regions (T-UCRs) and large intergenic non-coding RNAs (lincRNAs) are emerging as key elements of cellular homeostasis. Along with microRNAs, dysregulation of these ncRNAs is being found to have relevance not only to tumorigenesis, but also to neurological, cardiovascular, developmental and other diseases. There is great interest in therapeutic strategies to counteract these perturbations of ncRNAs.

Genomic Safe Harbors in Human iPS Cells

Abstract SCI-47Current hematopoietic stem cell (HSC) gene therapy relies on randomly integrated retroviral vectors and is hampered by the risk of insertional oncogenesis often leading to leukemia. This risk would be minimized if therapeutic transgenes could be inserted in selected sites of the genome that permit appropriate function without disruption or dysregulation of endogenous genes — referred to as “genomic safe harbors.” The advent of induced pluripotent stem (iPS) cell technology offered unprecedented opportunities for the genetic engineering of human cells. iPS cells, unlike HSC, can be extensively cultured in vitro, enabling the selection and study of unique sites of transgene integration for the first time in a relevant setting. We proposed a definition of safe harbor sites, based on their topology in the genome with relation to coding genes and other genomic landmarks, using five criteria: (i) distance of at least 50 kb from the 5' end of any gene, (ii) distance of at least 300 kb from any cancer-related gene, (iii) distance of at least 300 kb from any microRNA, (iv) location outside a transcription unit, and (v) location outside ultraconserved regions of the human genome (1). To test them, we developed a strategy to select iPS cell clones harboring a single copy of a randomly integrating vector at sites that meet our safe harbor criteria. In a recent proof-of-principle study, using a model for genetic correction of β-thalassemia major, we demonstrated that erythroid progeny of patient-specific iPS cell clones harboring a lentivirally encoded β-globin transgene in a safe harbor site express therapeutic levels of β-globin without perturbing neighboring genes. This approach, entailing the prospective screening and selection of integration sites, based on combined bioinformatics and functional analyses, provides a robust and dependable strategy for the genetic engineering of human iPS cells. iPS cell-derived cell products used in regenerative medicine will need to be genetically engineered to correct a genetic disease or permit in vivo cell tracking or the elimination of residual undifferentiated cells or progeny gone astray. Our strategy should be broadly applicable to introducing reporter, suicide, or therapeutic genes in a clinically relevant manner. We are currently exploiting this strategy to express a conditional HSV-tk suicide gene for purging of iPS cell progeny from teratoma-initiating cells. With the emergence of improved technologies for homologous recombination into human cells, targeted gene addition may soon become a realistic option if predefined validated safe harbor sites in the human genome are identified. We are utilizing our selection strategy in iPS cells (using lentiviral vectors with reporter cassettes that can be exchanged using Cre recombinase-mediated cassette exchange) as a platform for the de novo discovery and characterization of putative universal safe harbor sites that can be broadly used for the genetic engineering of multiple human cell types. Disclosures:No relevant conflicts of interest to declare.

I-ADHoRe 3.0—fast and sensitive detection of genomic homology in extremely large data sets

Comparative genomics is a powerful means to gain insight into the evolutionary processes that shape the genomes of related species. As the number of sequenced genomes increases, the development of software to perform accurate cross-species analyses becomes indispensable. However, many implementations that have the ability to compare multiple genomes exhibit unfavorable computational and memory requirements, limiting the number of genomes that can be analyzed in one run. Here, we present a software package to unveil genomic homology based on the identification of conservation of gene content and gene order (collinearity), i-ADHoRe 3.0, and its application to eukaryotic genomes. The use of efficient algorithms and support for parallel computing enable the analysis of large-scale data sets. Unlike other tools, i-ADHoRe can process the Ensembl data set, containing 49 species, in 1 h. Furthermore, the profile search is more sensitive to detect degenerate genomic homology than chaining pairwise collinearity information based on transitive homology. From ultra-conserved collinear regions between mammals and birds, by integrating coexpression information and protein–protein interactions, we identified more than 400 regions in the human genome showing significant functional coherence. The different algorithmical improvements ensure that i-ADHoRe 3.0 will remain a powerful tool to study genome evolution.

Screening and cell-based assessment of mutations in the Aristaless-related homeobox (ARX) gene

ARX mutations cause a diverse spectrum of human disorders, ranging from severe brain and genital malformations to non-syndromic intellectual disability (ID). ARX is a transcription factor with multiple domains that include four polyalanine (pA) tracts, the first two of which are frequently expanded by mutations. We progressively screened DNA samples from 613 individuals with ID initially for the most frequent ARX mutations (c.304ins(GCG)(7)'expansion' of pA1 and c.429_452dup 'dup24bp' of pA2). Five hundred samples without pA1 or pA2 mutations had the entire ARX ORF screened by single stranded polymorphism conformation (SSCP) and/or denaturing high pressure liquid chromatography (dHPLC) analysis. Overall, eight families with six mutations in ARX were identified (1.31%): five duplication mutations in pA2 (0.82%) with three new clinical reports of families with the dup24bp and two duplications larger than the dup24bp mutation discovered (dup27bp, dup33bp); and three point mutations (0.6%), including one novel mutation in the homeodomain (c.1074G>T). Four ultraconserved regions distal to ARX (uc466-469) were also screened in a subset of 94 patients, with three unique nucleotide changes identified in two (uc466, uc467). The subcellular localization of full length ARX proteins was assessed for 11 variants. Protein mislocalization increased as a function of pA2 tract length and phenotypic severity, as has been previously suggested for pA1. Similarly, protein mislocalization of the homeodomain mutations also correlated with clinical severity, suggesting an emerging genotype vs cellular phenotype correlation.