Genes implicated by a methylome-wide schizophrenia study in neonatal blood show differential expression in adult brain samples.

Schizophrenia is a disabling disorder involving genetic predisposition in combination with environmental influences that likely act via dynamic alterations of the epigenome and the transcriptome but its detailed pathophysiology is largely unknown. We performed cell-type specific methylome-wide association study of neonatal blood (N = 333) from individuals who later in life developed schizophrenia and controls. Suggestively significant associations (P < 1.0 × 10-6) were detected in all cell-types and in whole blood with methylome-wide significant associations in monocytes (P = 2.85 × 10-9-4.87 × 10-9), natural killer cells (P = 1.72 × 10-9-7.82 × 10-9) and B cells (P = 3.8 × 10-9). Validation of methylation findings in post-mortem brains (N = 596) from independent schizophrenia cases and controls showed significant enrichment of transcriptional differences (enrichment ratio = 1.98-3.23, P = 2.3 × 10-3-1.0 × 10-5), with specific highly significant differential expression for, for example, BDNF (t = -6.11, P = 1.90 × 10-9). In addition, expression difference in brain significantly predicted schizophrenia (multiple correlation = 0.15-0.22, P = 3.6 × 10-4-4.5 × 10-8). In summary, using a unique design combining pre-disease onset (neonatal) blood methylomic data and post-disease onset (post-mortem) brain transcriptional data, we have identified genes of likely functional relevance that are associated with schizophrenia susceptibility, rather than confounding disease associated artifacts. The identified loci may be of clinical value as a methylation-based biomarker for early detection of increased schizophrenia susceptibility.

Circulating miRNAs as Potential Biomarkers for Patient Stratification in Bipolar Disorder: A Combined Review and Data Mining Approach.

Bipolar disorder is a debilitating psychiatric condition that is shaped in a concerted interplay between hereditary and triggering risk factors. Profound depression and mania define the disorder, but high clinical heterogeneity among patients complicates diagnosis as well as pharmacological intervention. Identification of peripheral biomarkers that capture the genomic response to the exposome may thus progress the development of personalized treatment. MicroRNAs (miRNAs) play a prominent role in of post-transcriptional gene regulation in the context of brain development and mental health. They are coordinately modulated by multifarious effectors, and alteration in their expression profile has been reported in a variety of psychiatric conditions. Intriguingly, miRNAs can be released from CNS cells and enter circulatory bio-fluids where they remain remarkably stable. Hence, peripheral circulatory miRNAs may act as bio-indicators for the combination of genetic risk, environmental exposure, and/or treatment response. Here we provide a comprehensive literature search and data mining approach that summarize current experimental evidence supporting the applicability of miRNAs for patient stratification in bipolar disorder.

Comparison of electrostatic and mechanical cell sorting with limited starting material.

Isolation of multiple cell populations from limited starting material and with minimal influence on cell homeostasis and viability are common requirements in both basic and clinical research. Fluorescence-activated cell sorting (FACS) is the most commonly applied sorting methodology with the majority of instruments being based on high pressure and electrostatic deflection. A more recent technology is based on a mechanical valve, operating at low pressure. In the present work we compared the two technologies by parallel sorting of small amounts of peripheral blood and umbilical cord blood on a BD FACSAria™ III and Miltenyi MACSQuant® Tyto® instrument. Concurrent manually performed magnetic-based cell sorting served as reference. Sorting metrics, including purity and viability, were compared. Expression of the heat-shock protein HSPA1A immediately post sorting and the proliferation potential of sorted T-cells in vitro was assessed. In general, there was little to distinguish the two fluorescence-activated technologies with regard to sorting metrics and HSPA1A expression. Variation, however, with respect to recovery and viability, was much smaller among Tyto sorted samples. The proliferation potential of Tyto-sorted T-cells was significantly higher compared to Aria-sorted T-cells, indicating that T-cells of the Tyto instrument are less perturbed. In summary, cell types of blood origin including CD34+ cells could effectively be isolated from small input amounts with either fluorescence-activated technology with little immediate effect on viability. The mechanical valve-based sorting by the Tyto instrument; however, appeared to perturb the cells to a lesser extent as judged by their proliferation potential.

Neuropsin in mental health.

Neuropsin is a brain-expressed extracellular matrix serine protease that governs synaptic plasticity through activity-induced proteolytic cleavage of synaptic proteins. Its substrates comprise several molecules central to structural synaptic plasticity, and studies in rodents have documented its role in cognition and the behavioral and neurobiological response to stress. Intriguingly, differential usage of KLK8 (neuropsin gene) splice forms in the fetal and adult brain has only been reported in humans, suggesting that neuropsin may serve a specialized role in human neurodevelopment. Through systematic interrogation of large-scale genetic data, we review KLK8 regulation in the context of mental health and provide a summary of clinical and preclinical evidence supporting a role for neuropsin in the pathogenesis of mental illness.

Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples.

We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.

Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots.

BACKGROUND: In utero and early-life experienced environmental exposures are suggested to play an important role in many multifactorial diseases potentially mediated through lasting effects on the epigenome. As the epigenome in addition remains modifiable throughout life, identifying specific disease-relevant biomarkers may prove challenging. This has led to an increased interest in epigenome-wide association studies using dried blood spots (DBS) routinely collected in perinatal screening programs. Such programs are in place in numerous countries around the world producing large and unique biobanks. However, availability of this biological material is highly limited as each DBS is made only from a few droplets of blood and storage conditions may be suboptimal for epigenetic studies. Furthermore, as relevant markers may reside outside gene bodies, epigenome-wide interrogation is needed. RESULTS: Here we demonstrate, as a proof of principle, that genome-wide interrogation of the methylome based on methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-seq) is feasible using a single 3.2 mm DBS punch (60 ng DNA) from filter cards archived for up to 16 years. The enrichment profile, sequence quality and distribution of reads across genetic regions were comparable between samples archived 16 years, 4 years and a freshly prepared control sample. CONCLUSIONS: In summary, we show that high-quality MeDIP-seq data is achievable from neonatal screening filter cards stored at room temperature, thereby providing information on annotated as well as on non-RefSeq genes and repetitive elements. Moreover, the quantity of DNA from one DBS punch proved sufficient allowing for multiple epigenome studies using one single DBS.

Hypomethylation of FAM63B in bipolar disorder patients.

Bipolar disorder (BD) and schizophrenia (SZ) are known to share common genetic and psychosocial risk factors. A recent epigenome-wide association study performed on blood samples from SZ patients found significant hypomethylation of FAM63B in exon 9. Here, we used iPLEX-based methylation analysis to investigate two CpG sites in FAM63B in blood samples from 459 BD cases and 268 controls. Both sites were significantly hypomethylated in BD cases (lowest p value = 3.94 × 10(-8)). The methylation levels at the two sites were correlated, and no strong correlation was found with nearby single nucleotide polymorphisms (SNPs), suggesting that methylation differences at these sites are not readably picked up by genome-wide association studies. Overall, FAM63B hypomethylation was found in BD patients, thus replicating the initial finding in SZ patients. This study suggests that FAM63B is a shared epigenetic risk gene for the two disorders.

Generation of outbred Ace2 knockout mice by RNA transfection of TALENs displaying colitis reminiscent pathophysiology and inflammation.

The angiotensin I converting enzyme 2 (ACE2) is a key factor in the maintenance of intestinal homeostasis. Dysregulation of homeostasis can lead to inflammation of the colon (colitis), which can cause life-threatening enfeeblement or even cancer. Animal models are valuable surrogates in deciphering the pathology behind such human conditions and for screening of putative therapeutic targets or treatment paradigms. However, development of disease models can be time-consuming and technical demanding, which might hamper their application-value. In this study, we genetically disrupted the mouse Ace2 gene by direct injection of in vitro transcribed mRNA coding for transcription activator-like effector nucleases (TALENs) into the cytoplasm of outbred Kunming mouse zygotes. Consequently, somatic mutations were induced with an efficiency of 57%, of which 39% were frameshift mutations. Moreover, all modifications were stably transferred during germline transmission. In Ace2-knockout male mice (Ace2(-/y)), we observed severe chemical induced colitis, characterized by considerable weight loss, diarrhea and a shortened colon length. Histologically, Ace2 mutations resulted in the infiltration of leukocytes and the overt damage of the intestinal mucosal barrier. In addition, we detected an increased expression of inflammatory cytokines in the colon tissue of Ace2(-/y) mice. Collectively, the data indicate that high targeting efficiency and heritability can be achieved in an outbred mouse model by zygote injection of TALEN mRNA. Furthermore, the generated Ace2(-/y) mice display phenotypic traits reminiscent of colitis and we anticipate that such mice can be of value in studies of the intestinal microbiome or fecal transplantation.

DNA transposition by protein transduction of the piggyBac transposase from lentiviral Gag precursors.

DNA transposon-based vectors have emerged as gene vehicles with a wide biomedical and therapeutic potential. So far, genomic insertion of such vectors has relied on the co-delivery of genetic material encoding the gene-inserting transposase protein, raising concerns related to persistent expression, insertional mutagenesis and cytotoxicity. This report describes potent DNA transposition achieved by direct delivery of transposase protein. By adapting integrase-deficient lentiviral particles (LPs) as carriers of the hyperactive piggyBac transposase protein (hyPBase), we demonstrate rates of DNA transposition that are comparable with the efficiency of a conventional plasmid-based strategy. Embedded in the Gag polypeptide, hyPBase is robustly incorporated into LPs and liberated from the viral proteins by the viral protease during particle maturation. We demonstrate lentiviral co-delivery of the transposase protein and vector RNA carrying the transposon sequence, allowing robust DNA transposition in a variety of cell types. Importantly, this novel delivery method facilitates a balanced cellular uptake of hyPBase, as shown by confocal microscopy, and allows high-efficiency production of clones harboring a single transposon insertion. Our findings establish engineered LPs as a new tool for transposase delivery. We believe that protein transduction methods will increase applicability and safety of DNA transposon-based vector technologies.

Integrase-defective lentiviral vectors--a stage for nonviral integration machineries.

Gene vehicles derived from lentiviruses have become highly esteemed tools for gene transfer and genomic insertion in a wealth of cell types both in vivo and ex vivo. However, accumulating evidence of preferred insertion into actively transcribed genes, driven by biological properties of the parental human immunodeficiency virus type 1, has questioned the safety of this vector technology. As a consequence, integrase-defective lentiviral vectors [IDLVs], carrying an inactive integrase protein, have been developed and used with success for persistent in vivo gene transfer to quiescent or slowly dividing cells. We and others have shown that episomal DNA delivered by IDLVs may serve as a substrate for heterologous integration machineries, including recombinases and transposases, and homologous recombination triggered by nuclease-induced DNA damage. New vector systems that combine the best of lentiviral gene delivery and nonviral integration systems are under development. The first prototypes of such hybrid lentiviral vectors facilitate efficient gene transfer and show profiles of insertion that are not dictated by the biological constraints of the normal integration pathway and are, therefore, significantly different from the profile of conventional lentiviral vectors. The stage is set for further exploration of these vectors. In this review, we summarize the background and short history of hybrid IDLV-based vector systems and discuss their applicability in gene therapy and treatment of genetic disease.

A Sleeping Beauty DNA transposon-based genetic sensor for functional screening of vitamin D3 analogues.

BACKGROUND: Analogues of vitamin D3 are extensively used in the treatment of various illnesses, such as osteoporosis, inflammatory skin diseases, and cancer. Functional testing of new vitamin D3 analogues and formulations for improved systemic and topical administration is supported by sensitive screening methods that allow a comparative evaluation of drug properties. As a new tool in functional screening of vitamin D3 analogues, we describe a genomically integratable sensor for sensitive drug detection. This system facilitates assessment of the pharmacokinetic and pharmadynamic properties of vitamin D3 analogues. The tri-cistronic genetic sensor encodes a drug-sensoring protein, a reporter protein expressed from an activated sensor-responsive promoter, and a resistance marker. RESULTS: The three expression cassettes, inserted in a head-to-tail orientation in a Sleeping Beauty DNA transposon vector, are efficiently inserted as a single genetic entity into the genome of cells of interest in a reaction catalyzed by the hyperactive SB100X transposase. The applicability of the sensor for screening purposes is demonstrated by the functional comparison of potent synthetic analogues of vitamin D3 designed for the treatment of psoriasis and cancer. In clones of human keratinocytes carrying from a single to numerous insertions of the vitamin D3 sensor, a sensitive sensor read-out is detected upon exposure to even low concentrations of vitamin D3 analogues. In comparative studies, the sensor unveils superior potency of new candidate drugs in comparison with analogues that are currently in clinical use. CONCLUSIONS: Our findings demonstrate the use of the genetic sensor as a tool in first-line evaluation of new vitamin D3 analogues and pave the way for new types of drug delivery studies in sensor-transgenic animals.

Hybrid lentivirus-transposon vectors with a random integration profile in human cells.

Gene delivery by human immunodeficiency virus type 1 (HIV-1)-based lentiviral vectors (LVs) is efficient, but genomic integration of the viral DNA is strongly biased toward transcriptionally active loci resulting in an increased risk of insertional mutagenesis in gene therapy protocols. Nonviral Sleeping Beauty (SB) transposon vectors have a significantly safer insertion profile, but efficient delivery into relevant cell/tissue types is a limitation. In an attempt to combine the favorable features of the two vector systems we established a novel hybrid vector technology based on SB transposase-mediated insertion of lentiviral DNA circles generated during transduction of target cells with integrase (IN)-defective LVs (IDLVs). By construction of a lentivirus-transposon hybrid vector allowing transposition exclusively from circular viral DNA substrates, we demonstrate that SB transposase added in trans directs efficient transposon mobilization from DNA circles in vector-transduced cells. Both transfected plasmid DNA and transduced IDLVs can serve as the source of active transposase. Most important, we demonstrate that the SB transposase overrides the natural lentiviral integration pathway and directs vector integration less frequently toward transcriptional units, resulting in a random genomic integration profile. The novel hybrid vector system combines the attractive features of efficient gene delivery by viral transduction and a safer genomic integration profile by DNA transposition.

Genomic insertion of lentiviral DNA circles directed by the yeast Flp recombinase.

BACKGROUND: Circular forms of viral genomic DNA are generated during infection of cells with retroviruses like HIV-1. Such circles are unable to replicate and are eventually lost as a result of cell division, lending support to the prevalent notion that episomal retroviral DNA forms are dead-end products of reverse transcription. RESULTS: We demonstrate that circular DNA generated during transduction with HIV-1-based lentiviral vectors can be utilized as substrate for gene insertion directed by nonviral recombinases co-expressed in the target cells. By packaging of lentiviral genomic RNA in integrase-defective lentiviral vectors, harboring an inactive form of the viral integrase, the normal pathway for viral integration is blocked and circular vector DNA accumulates in transduced cells as a result. We find that the amount of DNA circles is increased 4-fold in cells transduced with integration-defective vectors relative to cells treated with integrase-proficient vectors. By transduction of target cells harboring engineered recognition sites for the yeast Flp recombinase with integration-defective lentiviral vectors containing an ATG-deficient hygromycin B selection gene we demonstrate precise integration of lentiviral vector-derived DNA circles in a drug-selective approach. Moreover, it is demonstrated that trans-acting Flp recombinase can be delivered by Flp-encoding transfected plasmid DNA or, alternatively, by co-transduced integrase-defective lentiviral vectors carrying a Flp expression cassette. CONCLUSION: Our data provide proof-of-principle that nonviral recombinases, like Flp, produced by plasmid DNA or non-integrating lentiviral vectors can gain access to circular viral recombination substrates and facilitate site-directed genomic insertion of such episomal DNA forms. Replacement of the normal viral integration machinery with nonviral mediators of integration represents a new platform for creation of lentiviral vectors with an altered integration profile.