Ten years of infant mental health in the Netherlands:Who are the clients?

Background: Infant Mental Health (IMH) is a topic of current interest that emerged over the past decades, concerned with alleviating suffering and enhancing the social and emotional competence of young children. Worldwide there is increasing recognition of infant psychopathology meriting intervention. However, there are still limited data regarding the prevalence of psychiatric disorders and sociodemographic characteristics of these youngest of patients in clinical settings. Aim: This large, descriptive study aims at presenting the socio-demographic and clinical characteristics of infants referred consecutively to three outpatient Infant Mental Health teams in the Netherlands between September 2000 and July 2013. Methods: The medical records of 783 infants were retrospectively examined and the data were collected from paper and electronic patient files. Clinical and socio-demographic characteristics were categorized in child factors, developmental milestones, family factors and clinical outcome measures (DSM-IV, DC:0-3R, WIPPSI-III, SON-R 2½-7). Results: Our sample showed significantly more boys (543, 69%) than girls (240, 31%) being referred to the Infant Mental Health teams. Most children were referred when they were four or five years of age, both boys and girls. Mean duration of treatment was about a year and a half (20.34 months, SD 18.87) and most reported diagnoses were ADHD/behavioral disorders, ASS and disorder in infancy/childhood NOS. Familial psychiatric disorders were reported in 242 families (41%). These findings are discussed in the light of earlier research

Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9.

The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease

Repair at Single Targeted DNA Double-Strand Breaks in Pluripotent and Differentiated Human Cells

Differences in ex vivo cell culture conditions can drastically affect stem cell physiology. We sought to establish an assay for measuring the effects of chemical, environmental, and genetic manipulations on the precision of repair at a single DNA double-strand break (DSB) in pluripotent and somatic human cells. DSBs in mammalian cells are primarily repaired by either homologous recombination (HR) or nonhomologous end-joining (NHEJ). For the most part, previous studies of DSB repair in human cells have utilized nonspecific clastogens like ionizing radiation, which are highly nonphysiologic, or assayed repair at randomly integrated reporters. Measuring repair after random integration is potentially confounded by locus-specific effects on the efficiency and precision of repair. We show that the frequency of HR at a single DSB differs up to 20-fold between otherwise isogenic human embryonic stem cells (hESCs) based on the site of the DSB within the genome. To overcome locus-specific effects on DSB repair, we used zinc finger nucleases to efficiently target a DSB repair reporter to a safe-harbor locus in hESCs and a panel of somatic human cell lines. We demonstrate that repair at a targeted DSB is highly precise in hESCs, compared to either the somatic human cells or murine embryonic stem cells. Differentiation of hESCs harboring the targeted reporter into astrocytes reduces both the efficiency and precision of repair. Thus, the phenotype of repair at a single DSB can differ based on either the site of damage within the genome or the stage of cellular differentiation. Our approach to single DSB analysis has broad utility for defining the effects of genetic and environmental modifications on repair precision in pluripotent cells and their differentiated progeny

Genome Res

Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a "safe harbor" locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells

SplitAx:A novel method to assess the function of engineered nucleases

Engineered nucleases have been used to generate knockout or reporter cell lines and a range of animal models for human disease. These new technologies also hold great promise for therapeutic genome editing. Current methods to evaluate the activity of these nucleases are time consuming, require extensive optimization and are hampered by readouts with low signals and high background. We have developed a simple and easy to perform method (SplitAx) that largely addresses these issues and provides a readout of nuclease activity. The assay involves splitting the N-terminal (amino acid 1-158) coding region of GFP and an out-of-frame of C-terminal region with a nuclease binding site sequence. Following exposure to the test nuclease, cutting and repair by error prone non-homologous end joining (NHEJ) restores the reading frame resulting in the production of a full length fluorescent GFP protein. Fluorescence can also be restored by complementation between the N-terminal and C-terminal coding sequences in trans. We demonstrate successful use of the SplitAx assay to assess the function of zinc finger nucleases, CRISPR hCAS9 and TALENS. We also test the activity of multiple gRNAs in CRISPR/hCas9/D10A systems. The zinc finger nucleases and guide RNAs that showed functional activity in the SplitAx assay were then used successfully to target the endogenous AAVS1, SOX6 and Cfms loci. This simple method can be applied to other unrelated proteins such as ZsGreen1 and provides a test system that does not require complex optimization

TLE4 regulation of wnt-mediated inflammation underlies its role as a tumor suppressor in myeloid leukemia

The presence of AML1-ETO (RUNX1-CBF2T1), a fusion oncoprotein resulting from a t(8;21) chromosomal translocation, has been implicated as a necessary but insufficient event in the development of a subset of acute myeloid leukemias (AML). While AML1-ETO prolongs survival and inhibits differentiation of hematopoietic stem cells (HSC), other contributory events are needed for cell proliferation and leuke-mogenesis. We have postulated that specific tumor suppressor genes keep the leukemic potential of AML1-ETO in check. In studying del(9q), one of the most common concomitant chromosomal abnormalities with t(8;21), we identified the loss of an apparent tumor suppressor, TLE4, that appears to cooperate with AML1-ETO to confer a leukemic phenotype. This study sought to identify the molecular basis of this cooperation. We show that the loss of TLE4 confers proliferative advantage to leukemic cells, simultaneous with an upregulation of a pro-inflammatory signature mediated through aberrant increases in Wnt signaling activity. We further demonstrate that inhibition of cyclooxygenase (COX) activity partly reverses the pro-leukemic phenotype due to TLE4 knockdown, pointing towards a novel therapeutic approach for myeloid leukemia

Functional genomics, proteomics, and regulatory DNA analysis in isogenic settings using zinc finger nuclease-driven transgenesis into a safe harbor locus in the human genome

Isogenic settings are routine in model organisms, yet remain elusive for genetic experiments on human cells. We describe the use of designed zinc finger nucleases (ZFNs) for efficient transgenesis without drug selection into the PPP1R12C gene, a “safe harbor” locus known as AAVS1. ZFNs enable targeted transgenesis at a frequency of up to 15% following transient transfection of both transformed and primary human cells, including fibroblasts and hES cells. When added to this locus, transgenes such as expression cassettes for shRNAs, small-molecule-responsive cDNA expression cassettes, and reporter constructs, exhibit consistent expression and sustained function over 50 cell generations. By avoiding random integration and drug selection, this method allows bona fide isogenic settings for high-throughput functional genomics, proteomics, and regulatory DNA analysis in essentially any transformed human cell type and in primary cells