Invasive Assessment of Microvascular Resistance in Hypertrophic Cardiomyopathy With Echocardiographic Correlates.

INTRODUCTION: Hypertrophic cardiomyopathy (HCM) is often associated with ischaemia despite lack of focal epicardial coronary stenosis. Our aim was to assess invasive coronary microvascular circulation and correlate findings with echocardiography. METHODS: We prospectively enrolled patients with HCM and controls who were referred for diagnostic coronary angiography. A pressure-temperature sensor coronary guidewire was used with intracoronary injections of room-temperature saline to measure mean coronary transit time during rest and hyperaemia induced with intravenous adenosine. The index of microvascular resistance (IMR) was calculated. Left ventricular mass was calculated during echocardiographic studies. RESULTS: Patients with HCM (n=12) and controls (n=7), had similar demographics. Left ventricular ejection fraction was higher in HCM (76.7%±11.0% vs 55.0%±15.9%, p=0.003). IMR was non-significantly higher in HCM (21.7±10.2 vs 15.3±4.8, p=0.16). Only patients with HCM had abnormal IMR (>25). Coronary flow reserve was non-significantly higher in HCM (2.7±1.6 vs 2.1±1.2, p=0.34). IMR correlated with left ventricular mass in hypertrophic cardiomyopathy subjects (Pearson r=0.68, p=0.02). CONCLUSIONS: Microvascular dysfunction as assessed by IMR may be abnormal in HCM and is correlated with left ventricular mass.

The impact of interventions for youth experiencing homelessness on housing, mental health, substance use, and family cohesion: a systematic review.

BACKGROUND: Youth often experience unique pathways into homelessness, such as family conflict, child abuse and neglect. Most research has focused on adult homeless populations, yet youth have specific needs that require adapted interventions. This review aims to synthesize evidence on interventions for youth and assess their impacts on health, social, and equity outcomes. METHODS: We systematically searched Medline, Embase, PsycINFO, and other databases from inception until February 9, 2018 for systematic reviews and randomized controlled trials on youth interventions conducted in high income countries. We screened title and abstract and full text for inclusion, and data extraction were completed in duplicate, following the PRISMA-E (equity) review approach. RESULTS: Our search identified 11,936 records. Four systematic reviews and 18 articles on randomized controlled trials met the inclusion criteria. Many studies reported on interventions including individual and family therapies, skill-building, case management, and structural interventions. Cognitive behavioural therapy led to improvements in depression and substance use, and studies of three family-based therapies reported decreases in substance use. Housing first, a structural intervention, led to improvements in housing stability. Many interventions showed inconsistent results compared to services as usual or other interventions, but often led to improvements over time in both the intervention and comparison group. The equity analysis showed that equity variables were inconsistently measured, but there was data to suggest differential outcomes based upon gender and ethnicity. CONCLUSIONS: This review identified a variety of interventions for youth experiencing homelessness. Promising interventions include cognitive behavioural therapy for addressing depression, family-based therapy for substance use outcomes, and housing programs for housing stability. Youth pathways are often unique and thus prevention and treatment may benefit from a tailored and flexible approach.

Regulation of SPRY3 by X chromosome and PAR2-linked promoters in an autism susceptibility region.

Sprouty proteins are regulators of cell growth and branching morphogenesis. Unlike mouse Spry3, which is X-linked, human SPRY3 maps to the pseudoautosomal region 2; however, the human Y-linked allele is not expressed due to epigenetic silencing by an unknown mechanism. SPRY3 maps adjacent to X-linked Trimethyllysine hydroxylase epsilon (TMLHE), recently identified as an autism susceptibility gene. We report that Spry3 is highly expressed in central and peripheral nervous system ganglion cells in mouse and human, including cerebellar Purkinje cells and retinal ganglion cells. Transient over-expression or knockdown of Spry3 in cultured mouse superior cervical ganglion cells inhibits and promotes, respectively, neurite growth and branching. A 0.7 kb gene fragment spanning the human SPRY3 transcriptional start site recapitulates the endogenous Spry3-expression pattern in LacZ reporter mice. In the human and mouse the SPRY3 promoter contains an AG-rich repeat and we found co-expression, and promoter binding and/or regulation of SPRY3 expression by transcription factors MAZ, EGR1, ZNF263 and PAX6. We identified eight alleles of the human SPRY3 promoter repeat in Caucasians, and similar allele frequencies in autism families. We characterized multiple SPRY3 transcripts originating at two CpG islands in the X-linked F8A3-TMLHE region, suggesting X chromosome regulation of SPRY3. These findings provide an explanation for differential regulation of X and Y-linked SPRY3 alleles. In addition, the presence of a SPRY3 transcript exon in a previously described X chromosome deletion associated with autism, and the cerebellar interlobular variation in Spry3 expression coincident with the reported pattern of Purkinje cell loss in autism, suggest SPRY3 as a candidate susceptibility locus for autism.

The Impact of Peripheral Nerve Stimulation on Coronary Blood Flow and Endothelial Function.

PURPOSE: The geko™ device is a small transcutaneous nerve stimulator that is applied non-invasively to the skin over the common peroneal nerve to stimulate peripheral blood flow. The purpose of this study was to investigate the effect of peripheral nerve stimulation on coronary flow dynamics and systemic endothelial function. METHODS: We enrolled 10 male patients, age 59 ± 11 years, with symptomatic obstructive coronary disease undergoing percutaneous coronary intervention (PCI). Coronary flow dynamics were assessed invasively using Doppler flow wire at baseline and with nerve stimulation for 4 min. Measurements were taken in the stenotic coronary artery and in a control vessel without obstructive disease. At a separate visit, peripheral blood flow at the popliteal artery (using duplex ultrasound assessment) and endothelial function assessed by peripheral artery tonometry (PAT) were measured at baseline and after one hour of nerve stimulation. RESULTS: Compared to baseline, there was a significant increase in coronary blood flow as measured by average peak velocity (APV) in the control vessel with nerve stimulation (20.3 ± 7.7 to 23.5 ± 10 cm/s; p = 0.03) and non-significant increase in the stenotic vessel (21.9 ± 12 to 23.9 ± 12.9 cm/s; p = 0.23). Coronary flow reserve did not change significantly. Reactive hyperemia-peripheral arterial tonometry (Rh-PAT) increased from 2.28 ± 0.39 to 2.67 ± 0.6, p = 0.045. CONCLUSIONS: A few minutes of peripheral nerve stimulation may improve coronary blood flow. This effect is more prominent in non-stenotic vessels. Longer stimulation improved endothelial function.

Late-replicating heterochromatin is characterized by decreased cytosine methylation in the human genome.

Heterochromatin is believed to be associated with increased levels of cytosine methylation. With the recent availability of genome-wide, high-resolution molecular data reflecting chromatin organization and methylation, such relationships can be explored systematically. As well-defined surrogates for heterochromatin, we tested the relationship between DNA replication timing and DNase hypersensitivity with cytosine methylation in two human cell types, unexpectedly finding the later-replicating, more heterochromatic regions to be less methylated than early replicating regions. When we integrated gene-expression data into the study, we found that regions of increased gene expression were earlier replicating, as previously identified, and that transcription-targeted cytosine methylation in gene bodies contributes to the positive correlation with early replication. A self-organizing map (SOM) approach was able to identify genomic regions with early replication and increased methylation, but lacking annotated transcripts, loci missed in simple two variable analyses, possibly encoding unrecognized intergenic transcripts. We conclude that the relationship of cytosine methylation with heterochromatin is not simple and depends on whether the genomic context is tandemly repetitive sequences often found near centromeres, which are known to be heterochromatic and methylated, or the remaining majority of the genome, where cytosine methylation is targeted preferentially to the transcriptionally active, euchromatic compartment of the genome.

"Like an umbrella, protecting me from the rain until I get to my destination": Evaluating the implementation of a tailored primary care model for urban marginalized populations.

BACKGROUND: Improving health equity and access to the highest possible standard of health care is a key issue of social accountability. Centretown Community Health Centre in Ottawa, Canada has iteratively developed a program to target and serve marginalized and complex populations since 1999. The program implementation was evaluated using a validated implementation framework. METHODS: Quantitative and qualitative data were collected through a health records extraction (n = 570), a client complexity assessment tool (n = 74), semi-structured interviews with clients and key stakeholders (n = 41), and a structured client satisfaction survey (n = 30). Data were analyzed using descriptive statistics and inductive thematic analysis. RESULTS: Five hundred and seventy unique clients were seen between November 1-30, 2021. A third of clients (34%) did not have a provincial health card for access to universal health care services, and most (68%) were homeless or a resident of rooming houses. Most clients who reported their income (92%) were at or below Canada's official poverty line. The total mean complexity score for clients seen over a one-month period (n = 74) was 16.68 (SD 6.75) where a total score of at least 13 of 33 is perceived to be a threshold for client biopsychosocial complexity. Clients gained the majority of their total score from the Social support assessment component of the tool. Clients (n = 31) and key informants (n = 10) highlighted the importance of building relationships with this population, providing wrap-around care, and providing low-barrier care as major strength to the Urban Health program (UH). Key areas for improvement included the need to: i) increase staff diversity, ii) expand program hours and availability, and iii) improve access to harm reduction services. Clients appeared to be highly satisfied with the program, rating the program an average total score of 18.50 out of 20. CONCLUSIONS: The program appears to serve marginalized and complex clients and seems well-received by the community. Our findings have relevance for other health care organizations seeking to better serve marginalized and medically and socially complex individuals and families in their communities.

The Wasp System: an open source environment for managing and analyzing genomic data.

The challenges associated with the management, analysis and interpretation of assays based on massively-parallel sequencing (MPS) are both individually complex and numerous. We describe what we believe to be the appropriate solution, one that represents a departure from traditional computational biology approaches. The Wasp System is an open source, distributed package written in Spring/J2EE that creates a foundation for development of an end-to-end solution for MPS-based experiments or clinical tests. Recognizing that one group will be unable to solve these challenges in isolation, we describe a nurtured open source development model that will allow the software to be collectively used, shared and developed. The ultimate goal is to emulate resources such as the Virtual Observatory of the astrophysics community, enabling computationally-inexpert scientists and clinicians to explore and interpret their MPS data. Here we describe the current implementation and development of the Wasp System and the roadmap for its community development.

The Einstein Genome Gateway using WASP - a high throughput multi-layered life sciences portal for XSEDE.

Massively-parallel sequencing (MPS) technologies and their diverse applications in genomics and epigenomics research have yielded enormous new insights into the physiology and pathophysiology of the human genome. The biggest hurdle remains the magnitude and diversity of the datasets generated, compromising our ability to manage, organize, process and ultimately analyse data. The Wiki-based Automated Sequence Processor (WASP), developed at the Albert Einstein College of Medicine (hereafter Einstein), uniquely manages to tightly couple the sequencing platform, the sequencing assay, sample metadata and the automated workflows deployed on a heterogeneous high performance computing cluster infrastructure that yield sequenced, quality-controlled and 'mapped' sequence data, all within the one operating environment accessible by a web-based GUI interface. WASP at Einstein processes 4-6 TB of data per week and since its production cycle commenced it has processed ~ 1 PB of data overall and has revolutionized user interactivity with these new genomic technologies, who remain blissfully unaware of the data storage, management and most importantly processing services they request. The abstraction of such computational complexity for the user in effect makes WASP an ideal middleware solution, and an appropriate basis for the development of a grid-enabled resource - the Einstein Genome Gateway - as part of the Extreme Science and Engineering Discovery Environment (XSEDE) program. In this paper we discuss the existing WASP system, its proposed middleware role, and its planned interaction with XSEDE to form the Einstein Genome Gateway.

Loss of MMR and TGFBR2 Increases the Susceptibility to Microbiota-Dependent Inflammation-Associated Colon Cancer.

BACKGROUND AND AIMS: Mutations in DNA mismatch repair (MMR) genes are causative in Lynch syndrome and a significant proportion of sporadic colorectal cancers (CRCs). MMR-deficient (dMMR) CRCs display increased mutation rates, with mutations frequently accumulating at short repetitive DNA sequences throughout the genome (microsatellite instability). The TGFBR2 gene is one of the most frequently mutated genes in dMMR CRCs. Therefore, we generated an animal model to study how the loss of both TGFBR2 signaling impacts dMMR-driven intestinal tumorigenesis in vivo and explore the impact of the gut microbiota. METHODS: We generated VCMsh2/Tgfbr2 mice in which Msh2loxP and Tgfbr2loxP alleles are inactivated by Villin-Cre recombinase in the intestinal epithelium. VCMsh2/Tgfbr2 mice were analyzed for their rate of intestinal cancer development and for the mutational spectra and gene expression profiles of tumors. In addition, we assessed the impact of chemically induced chronic inflammation and gut microbiota composition on colorectal tumorigenesis. RESULTS: VCMsh2/Tgfbr2 mice developed small intestinal adenocarcinomas and CRCs with histopathological features highly similar to CRCs in Lynch syndrome patients. The CRCs in VCMsh2/Tgfbr2 mice were associated with the presence of colitis and displayed genetic and histological features that resembled inflammation-associated CRCs in human patients. The development of CRCs in VCMsh2/Tgfbr2 mice was strongly modulated by the gut microbiota composition, which in turn was impacted by the TGFBR2 status of the tumors. CONCLUSIONS: Our results demonstrate a synergistic interaction between MMR and TGFBR2 inactivation in inflammation-associated colon tumorigenesis and highlight the crucial impact of the gut microbiota on modulating the incidence of inflammation-associated CRCs.

Evidence-informed interventions and best practices for supporting women experiencing or at risk of homelessness: a scoping review with gender and equity analysis. / Interventions fondées sur des données probantes et pratiques exemplaires en matière de soutien apporté aux femmes en situation ou à risque d'itinérance : examen de la portée avec analyse fondée sur le sexe et l'équité.

INTRODUCTION: While much of the literature on homelessness is centred on the experience of men, women make up over one-quarter of Canada's homeless population. Research has shown that women experiencing homelessness are often hidden (i.e. provisionally housed) and have different pathways into homelessness and different needs as compared to men. The objective of this research is to identify evidence-based interventions and best practices to better support women experiencing or at risk of homelessness. METHODS: We conducted a scoping review with a gender and equity analysis. This involved searching MEDLINE, CINAHL, PsycINFO and other databases for systematic reviews and randomized trials, supplementing our search through reference scanning and grey literature, followed by a qualitative synthesis of the evidence that examined gender and equity considerations. RESULTS: Of the 4102 articles identified on homelessness interventions, only 4 systematic reviews and 9 randomized trials were exclusively conducted on women or published disaggregated data enabling a gender analysis. Interventions with the strongest evidence included post-shelter advocacy counselling for women experiencing homelessness due to intimate partner violence, as well as case management and permanent housing subsidies (e.g. tenant-based rental assistance vouchers), which were shown to reduce homelessness, food insecurity, exposure to violence and psychosocial distress, as well as promote school stability and child well-being. CONCLUSION: Much of the evidence on interventions to better support women experiencing homelessness focusses on those accessing domestic violence or family shelters. Since many more women are experiencing or at risk of hidden homelessness, population-based strategies are also needed to reduce gender inequity and exposure to violence, which are among the main structural drivers of homelessness among women.

Making every death count: institutional mortality accuracy at Ola During Children's Hospital, Sierra Leone.

INTRODUCTION: health care data accuracy feeds the development of sound healthcare policy and the prioritisation of interventions in scarce resource environments. We designed a retrospective study at the sole paediatric government hospital in Sierra Leone to examine mortality statistics, specifically: the accuracy of mortality data collected in 2017; and the quality of cause of death (CoD) reporting for 2017. METHODS: the retrospective audit included all available mortality statistics collected at the hospital during the 2017 calendar year. For the purpose of calculating a mortality rate, admission data was additionally gathered. Four different hospital entities were identified that collected mortality data (the Monitoring and Evaluation (M&E) office; the nurse ledgers; the office of births and deaths; and the mortuary). Data from each hospital entity were used for the comparative analysis. RESULTS: striking differences were found in the rate of hospital mortality reported by different entities. The M&E office (responsible for providing data to the ministry of health and sanitation) reported a hospital mortality rate of 2.94% in 2017. Mortuary and nursing admissions records showed a hospital mortality rate of 18.7%. Discrepancies and issues of quality in CoD reporting between hospital entities were identified. CONCLUSION: significant variations were found in the generation of official hospital mortality data. Mortality data informs health service prioritisation, resource distribution, outcome measures and epidemiological surveillance. Resources to support quality improvement initiatives are needed in the creation of an in-hospital system that reports accurate data with a process for real-time institutional data feedback.

Whole-Exome Sequencing (WES) for Illumina Short Read Sequencers Using Solution-Based Capture.

Next-generation sequencing (NGS) is transforming clinical research and diagnostics, vastly enhancing our ability to identify novel disease-causing genetic mutations and perform comprehensive diagnostic testing in the clinic. Whole-exome sequencing (WES) is a commonly used method which captures the majority of coding regions of the genome for sequencing, as these regions contain the majority of disease-causing mutations. The clinical applications of WES are not limited to diagnosis; the technique can be employed to help determine an optimal therapeutic strategy for a patient considering their mutation profile. WES may also be used to predict a patient's risk of developing a disease, e.g., type 2 diabetes (T2D), and can therefore be used to tailor advice for the patient about lifestyle choices that could mitigate those risks. Thus, genome sequencing strategies, such as WES, underpin the emerging field of personalized medicine. Initiatives also exist for sharing WES data in public repositories, e.g., the Exome Aggregation Consortium (ExAC) database. In time, by mining these valuable data resources, we will acquire a better understanding of the roles of both single rare mutations and specific combinations of common mutations (mutation signatures) in the pathology of complex diseases such as diabetes.Herein, we describe a protocol for performing WES on genomic DNA extracted from blood or saliva. Starting with gDNA extraction, we document preparation of a library for sequencing on Illumina instruments and the enrichment of the protein-coding regions from the library using the Roche NimbleGen SeqCap EZ Exome v3 kit; a solution-based capture method. We include details of how to efficiently purify the products of each step using the AMPure XP System and describe how to use qPCR to test the efficiency of capture, and thus determine finished library quality.

Lessons learned from expanded reproductive carrier screening in self-reported Ashkenazi, Sephardi, and Mizrahi Jewish patients.

BACKGROUND: Next-generation sequencing (NGS)-based panels have gained traction as a strategy for reproductive carrier screening. Their value for screening Ashkenazi Jewish (AJ) individuals, who have benefited greatly from population-wide targeted testing, as well as Sephardi/Mizrahi Jewish (SMJ) individuals (an underserved population), has not been fully explored. METHODS: The clinical utilization by 6,805 self-reported Jewish individuals of an expanded NGS panel, along with several ancillary assays, was assessed retrospectively. Data were extracted for a subset of 96 diseases that, during the panel design phase, were classified as being AJ-, SMJ-, or pan-Jewish/pan-ethnic-relevant. RESULTS: 64.6% of individuals were identified as carriers of one or more of these 96 diseases. Over 80% of the reported variants would have been missed by following recommended AJ screening guidelines. 10.7% of variants reported for AJs were in "SMJ-relevant genes," and 31.2% reported for SMJs were in "AJ-relevant genes." Roughly 2.5% of individuals carried a novel, likely pathogenic variant. One in 16 linked cohort couples was identified as a carrier couple for at least one of these 96 diseases. CONCLUSION: For maximal carrier identification, this study supports using expanded NGS panels for individuals of all Jewish backgrounds. This approach can better empower at-risk couples for reproductive decision making.

Conservation of pregnancy-specific glycoprotein (PSG) N domains following independent expansions of the gene families in rodents and primates.

BACKGROUND: Rodent and primate pregnancy-specific glycoprotein (PSG) gene families have expanded independently from a common ancestor and are expressed virtually exclusively in placental trophoblasts. However, within each species, it is unknown whether multiple paralogs have been selected for diversification of function, or for increased dosage of monofunctional PSG. We analysed the evolution of the mouse PSG sequences, and compared them to rat, human and baboon PSGs to attempt to understand the evolution of this complex gene family. RESULTS: Phylogenetic tree analyses indicate that the primate N domains and the rodent N1 domains exhibit a higher degree of conservation than that observed in a comparison of the mouse N1 and N2 domains, or mouse N1 and N3 domains. Compared to human and baboon PSG N domain exons, mouse and rat PSG N domain exons have undergone less sequence homogenisation. The high non-synonymous substitution rates observed in the CFG face of the mouse N1 domain, within a context of overall conservation, suggests divergence of function of mouse PSGs. The rat PSG family appears to have undergone less expansion than the mouse, exhibits lower divergence rates and increased sequence homogenisation in the CFG face of the N1 domain. In contrast to most primate PSG N domains, rodent PSG N1 domains do not contain an RGD tri-peptide motif, but do contain RGD-like sequences, which are not conserved in rodent N2 and N3 domains. CONCLUSION: Relative conservation of primate N domains and rodent N1 domains suggests that, despite independent gene family expansions and structural diversification, mouse and human PSGs retain conserved functions. Human PSG gene family expansion and homogenisation suggests that evolution occurred in a concerted manner that maintains similar functions of PSGs, whilst increasing gene dosage of the family as a whole. In the mouse, gene family expansion, coupled with local diversification of the CFG face, suggests selection both for increased gene dosage and diversification of function. Partial conservation of RGD and RGD-like tri-peptides in primate and rodent N and N1 domains, respectively, supports a role for these motifs in PSG function.

Structure and evolution of the mouse pregnancy-specific glycoprotein (Psg) gene locus.

BACKGROUND: The pregnancy-specific glycoprotein (Psg) genes encode proteins of unknown function, and are members of the carcinoembryonic antigen (Cea) gene family, which is a member of the immunoglobulin gene (Ig) superfamily. In rodents and primates, but not in artiodactyls (even-toed ungulates / hoofed mammals), there have been independent expansions of the Psg gene family, with all members expressed exclusively in placental trophoblast cells. For the mouse Psg genes, we sought to determine the genomic organisation of the locus, the expression profiles of the various family members, and the evolution of exon structure, to attempt to reconstruct the evolutionary history of this locus, and to determine whether expansion of the gene family has been driven by selection for increased gene dosage, or diversification of function. RESULTS: We collated the mouse Psg gene sequences currently in the public genome and expressed-sequence tag (EST) databases and used systematic BLAST searches to generate complete sequences for all known mouse Psg genes. We identified a novel family member, Psg31, which is similar to Psg30 but, uniquely amongst mouse Psg genes, has a duplicated N1 domain. We also identified a novel splice variant of Psg16 (bCEA). We show that Psg24 and Psg30 / Psg31 have independently undergone expansion of N-domain number. By mapping BAC, YAC and cosmid clones we described two clusters of Psg genes, which we linked and oriented using fluorescent in situ hybridisation (FISH). Comparison of our Psg locus map with the public mouse genome database indicates good agreement in overall structure and further elucidates gene order. Expression levels of Psg genes in placentas of different developmental stages revealed dramatic differences in the developmental expression profile of individual family members. CONCLUSION: We have combined existing information, and provide new information concerning the evolution of mouse Psg exon organization, the mouse Psg genomic locus structure, and the expression patterns of individual Psg genes. This information will facilitate functional studies of this complex gene family.

Exhaustive identification of human class II basic helix-loop-helix proteins by virtual library screening.

Cellular proliferation, specification and differentiation in developing tissues are tightly coordinated by groups of transcription factors in response to extrinsic and intrinsic signals. Furthermore, renewable pools of stem cells in adult tissues are subject to similar regulation. Basic helix-loop-helix (bHLH) proteins are a group of transcription factors that exert such a determinative influence on a variety of developmental pathways from C. elegans to humans, and we wished to exclusively identify novel members from within the whole human bHLH family. We have, therefore, developed an 'empirical custom fingerprint', to define the class II bHLH domain and exclusively identify these proteins in silico. We have identified nine previously uncharacterised human class II proteins, four of which were novel, by interrogating conceptual translations of the GenBank HTGS database. RT-PCR and mammalian 2-hybrid analysis of a subset of the factors demonstrated that they were indeed expressed, and were able to interact with an appropriate binding partner in vitro. Thus, we are now approaching an almost complete listing of human class II bHLH factors.

DNA demethylation by 5-aza-2'-deoxycytidine is imprinted, targeted to euchromatin, and has limited transcriptional consequences.

BACKGROUND: DNA methylation can be abnormally regulated in human disease and associated with effects on gene transcription that appear to be causally related to pathogenesis. The potential to use pharmacological agents that reverse this dysregulation is therefore an attractive possibility. To test how 5-aza-2'-deoxycytidine (5-aza-CdR) influences the genome therapeutically, we exposed non-malignant cells in culture to the agent and used genome-wide assays to assess the cellular response. RESULTS: We found that cells allowed to recover from 5-aza-CdR treatment only partially recover DNA methylation levels, retaining an epigenetic 'imprint' of drug exposure. We show very limited transcriptional responses to demethylation of not only protein-coding genes but also loci-encoding non-coding RNAs, with a limited proportion of the induced genes acquiring new promoter activation within gene bodies. The data revealed an uncoupling of DNA methylation effects at promoters, with demethylation mostly unaccompanied by transcriptional changes. The limited panel of genes induced by 5-aza-CdR resembles those activated in other human cell types exposed to the drug and represents loci targeted for Polycomb-mediated silencing in stem cells, suggesting a model for the therapeutic effects of the drug. CONCLUSIONS: Our results do not support the hypothesis of DNA methylation having a predominant role to regulate transcriptional noise in the genome and indicate that DNA methylation acts only as part of a larger complex system of transcriptional regulation. The targeting of 5-aza-CdR effects with its clastogenic consequences to euchromatin raises concerns that the use of 5-aza-CdR has innate tumorigenic consequences, requiring its cautious use in diseases involving epigenetic dysregulation.

An abundant placental transcript containing an IAP-LTR is allelic to mouse pregnancy-specific glycoprotein 23 (Psg23): cloning and genetic analysis.

Several families of endogenous retroviruses (ERVs) are expressed in mammalian placental tissues, and are implicated in aspects of placental development and function. We characterized the structure of abundant ERV-related transcripts in mouse placenta. In addition to the 7 kb full-length type I and 5 kb type I deleted intracisternal A-particle (IAP) transcripts, we identified and cloned an abundant 2 kb transcript encoding a novel member of the pregnancy-specific glycoprotein (Psg) gene family, which contains an IAP long terminal repeat (LTR) in the 3' untranslated region (UTR). The polyadenylation signal for the transcript is provided by the inserted LTR sequence. This sequence is allelic to Psg23 and is therefore denoted as Psg23(LTR). The transcript encodes a protein of 471 amino acids and has a domain organisation similar to previously described Psg proteins. Modelling of the protein N-domain produced a structure in good agreement with an existing crystalline structure for mouse sCEACAM1a. The LTR insertion is widely distributed among inbred mouse strains but is not found in 129/sv, CBA/2, or in wild mice. Cloning of the genomic region downstream of the LTR insertion site from the C57Bl/6J strain indicates that the insertion consists of a solo LTR without additional IAP sequence, and identified the original Psg23 polyadenylation signal sequence downstream of the insertion site. Psg23(LTR) was mapped to proximal chromosome 7 using the European collaborative interspecific mouse backcross (EUCIB) panel, and to yeast artificial chromosome (YAC) E072, which contains other members of the Psg gene family, by polymerase chain reaction (PCR). Northern blot analysis of RNA from adult and fetal mouse tissues and in situ hybridization to mid-gestation mouse embryos indicated that Psg23(LTR) is expressed predominantly in placental spongiotrophoblast. We detected a small, but statistically non-significant, bias in favour of transmission of Psg23(LTR) to the offspring of heterozygous parents. However, a larger study would be required to determine whether this allele is selectively advantageous to the developing embryo.