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Background: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occurs in a random manner in early development; however as tissues form, a 'patchiness' will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Determining the relationship between skewed XCI in different tissues and in different samples from the same tissue provides a molecular assessment of the developmental history of a particular tissue that can then be used to understand how genetic and epigenetic variation arises in development.Methods: XCI skewing was evaluated in and compared between amnion, chorion, trophoblast and mesenchyme using multiple sampling sites from 14 term placentae. XCI was also evaluated in chorionic villus samples obtained at multiple sites and depths from four additional term placentae. The pattern of variation was then compared with methylation variation associated with the H19/IGF2 imprinting control region (ICR); promoter regions of KISS1, PTPN6, CASP8 and APC; and LINE-1 elements.Results: Mean placental level of skewing for amnion and chorion are correlated, consistent with a common developmental origin of at least a component of these membranes from inner cell mass derivatives subsequent to XCI, while trophoblast appears to be derived independently, consistent with its origin from the trophectoderm. Villus samples taken from different depths spanning the fetal to maternal side of the placenta were highly clonally related. Comparing patterns of clonal growth identified through XCI to the distribution of epigenetic variation in other genomic regions suggests that some variation arises early in development (e.g. LINE-1 methylation), whereas other variation arises predominantly after villus tree formation (e.g. methylation at H19/IGF2 ICR).Conclusions: The patterns of XCI skewing are consistent with a model whereby each biopsied site of chorionic villi represents one or a few individual villus trees, each of which is clonally derived from only one or a few precursor cells. Sampling of placentae to evaluate changes associated with clinical pathology should be done with consideration of the tree-to-tree differences. A limitation of this study is the small number of placentas used and therefore placental-specific differences in variation could not be assessed. [ABSTRACT FROM AUTHOR]

Background: Fifteen years have passed since the outbreak of severe acute respiratory syndrome in Hong Kong. At that time, there were reports of heroic acts among professionals who cared for these patients, whose bravery and professionalism were highly praised. However, there are concerns about changes in new generation of nursing professionals., Objective: We aimed to examine the attitude of nursing students, should they be faced with severe acute respiratory syndrome patients during their future work., Research Design: A questionnaire survey was carried out to examine the attitude among final-year nursing students to three ethical areas, namely, duty of care, resource allocation, and collateral damage., Ethical Considerations: This study was carried out in accordance with the requirements and recommendations of the Central Research and Ethics Committee, School of Health Sciences at Caritas Institute of Higher Education., Findings: Complete responses from 102 subjects were analyzed. The overwhelming majority (96.1%) did not agree to participate in the intubation of severe acute respiratory syndrome patients if protective measures, that is, N95 mask and gown, were not available. If there were insufficient N95 masks for all the medical, nursing, and allied health workers in the hospital (resource allocation), 37.3% felt that the distribution of N95 masks should be by casting lot, while the rest disagreed. When asked about collateral damage, more than three-quarters (77.5%) said that severe acute respiratory syndrome patients should be admitted to intensive care unit. There was sex difference in nursing students' attitude toward severe acute respiratory syndrome care during pregnancy and influence of age in understanding intensive care unit care for these patients. Interestingly, 94.1% felt that there should be a separate intensive care unit for severe acute respiratory syndrome patients., Conclusion: As infection control practice and isolation facilities improved over the years, relevant knowledge and nursing ethical issues related to infectious diseases should become part of nursing education and training programs, especially in preparation for outbreaks of infectious diseases or distress.

Induced pluripotent stem cell (iPSC)-derived neurons are increasingly used to model Autism Spectrum Disorder (ASD), which is clinically and genetically heterogeneous. To study the complex relationship of penetrant and weaker polygenic risk variants to ASD, 'isogenic' iPSC-derived neurons are critical. We developed a set of procedures to control for heterogeneity in reprogramming and differentiation, and generated 53 different iPSC-derived glutamatergic neuronal lines from 25 participants from 12 unrelated families with ASD. Heterozygous de novo and rare-inherited presumed-damaging variants were characterized in ASD risk genes/loci. Combinations of putative etiologic variants ( GLI3/KIF21A or EHMT2/UBE2I ) in separate families were modeled. We used a multi-electrode array, with patch-clamp recordings, to determine a reproducible synaptic phenotype in 25% of the individuals with ASD (other relevant data on the remaining lines was collected). Our most compelling new results revealed a consistent spontaneous network hyperactivity in neurons deficient for CNTN5 or EHMT2. The biobank of iPSC-derived neurons and accompanying genomic data are available to accelerate ASD research., Editorial Note: This article has been through an editorial process in which authors decide how to respond to the issues raised during peer review. The Reviewing Editor's assessment is that all the issues have been addressed (see decision letter)., Competing Interests: ED, MF, SW, DR, SS, WW, AP, TT, JH, LC, VK, SW, PP, FR, RY, KS, JE No competing interests declared, SS Serves on the Scientific Advisory Committees of Population Bio and Deep Genomics, and intellectual property originating from his research and held at the Hospital for Sick Children is licensed to Lineagen, and separately Athena Diagnostics., (© 2019, Deneault et al.)

Contactin genes CNTN5 and CNTN6 code for neuronal cell adhesion molecules that promote neurite outgrowth in sensory-motor neuronal pathways. Mutations of CNTN5 and CNTN6 have previously been reported in individuals with autism spectrum disorders (ASDs), but very little is known on their prevalence and clinical impact. In this study, we identified CNTN5 and CNTN6 deleterious variants in individuals with ASD. Among the carriers, a girl with ASD and attention-deficit/hyperactivity disorder was carrying five copies of CNTN5. For CNTN6, both deletions (6/1534 ASD vs 1/8936 controls; P=0.00006) and private coding sequence variants (18/501 ASD vs 535/33480 controls; P=0.0005) were enriched in individuals with ASD. Among the rare CNTN6 variants, two deletions were transmitted by fathers diagnosed with ASD, one stop mutation CNTN6 W923X was transmitted by a mother to her two sons with ASD and one variant CNTN6 P770L was found de novo in a boy with ASD. Clinical investigations of the patients carrying CNTN5 or CNTN6 variants showed that they were hypersensitive to sounds (a condition called hyperacusis) and displayed changes in wave latency within the auditory pathway. These results reinforce the hypothesis of abnormal neuronal connectivity in the pathophysiology of ASD and shed new light on the genes that increase risk for abnormal sensory perception in ASD.

We are performing whole-genome sequencing of families with autism spectrum disorder (ASD) to build a resource (MSSNG) for subcategorizing the phenotypes and underlying genetic factors involved. Here we report sequencing of 5,205 samples from families with ASD, accompanied by clinical information, creating a database accessible on a cloud platform and through a controlled-access internet portal. We found an average of 73.8 de novo single nucleotide variants and 12.6 de novo insertions and deletions or copy number variations per ASD subject. We identified 18 new candidate ASD-risk genes and found that participants bearing mutations in susceptibility genes had significantly lower adaptive ability (P = 6 × 10 -4 ). In 294 of 2,620 (11.2%) of ASD cases, a molecular basis could be determined and 7.2% of these carried copy number variations and/or chromosomal abnormalities, emphasizing the importance of detecting all forms of genetic variation as diagnostic and therapeutic targets in ASD.

The Canadian beaver ( Castor canadensis ) is the largest indigenous rodent in North America. We report a draft annotated assembly of the beaver genome, the first for a large rodent and the first mammalian genome assembled directly from uncorrected and moderate coverage (< 30 ×) long reads generated by single-molecule sequencing. The genome size is 2.7 Gb estimated by k-mer analysis. We assembled the beaver genome using the new Canu assembler optimized for noisy reads. The resulting assembly was refined using Pilon supported by short reads (80 ×) and checked for accuracy by congruency against an independent short read assembly. We scaffolded the assembly using the exon-gene models derived from 9805 full-length open reading frames (FL-ORFs) constructed from the beaver leukocyte and muscle transcriptomes. The final assembly comprised 22,515 contigs with an N50 of 278,680 bp and an N50-scaffold of 317,558 bp. Maximum contig and scaffold lengths were 3.3 and 4.2 Mb, respectively, with a combined scaffold length representing 92% of the estimated genome size. The completeness and accuracy of the scaffold assembly was demonstrated by the precise exon placement for 91.1% of the 9805 assembled FL-ORFs and 83.1% of the BUSCO (Benchmarking Universal Single-Copy Orthologs) gene set used to assess the quality of genome assemblies. Well-represented were genes involved in dentition and enamel deposition, defining characteristics of rodents with which the beaver is well-endowed. The study provides insights for genome assembly and an important genomics resource for Castoridae and rodent evolutionary biology., (Copyright © 2017 Lok et al.)

A novel three-dimensional nanostructure based on cobalt phosphide nanoparticles (Co 2 P NPs) and heteroatom-doped mesoporous carbon spheres interconnected with graphene (3D PZM@Co 2 P@RGO) was facilely synthesized for the first time, and it was used for enhancing the flame retardancy and toxicity suppression of epoxy resins (EP) via a synergistic effect. Herein, the cross-linked polyphosphazene hollow spheres (PZM) were used as templates for the fabrication of 3D architecture. The 3D architecture based on Co 2 P-decorated heteroatom-doped carbon sphere and reduced graphene oxide was prepared via a carbonization procedure followed by a hydrothermal self-assembly strategy. The as-prepared material exhibits excellent catalytic activity with regard to the combustion process. Notably, inclusion of incorporating PZM@Co 2 P@RGO resulted in a dramatic reduction of the fire hazards of EP, such as a 47.9% maximum decrease in peak heat release rate and a 29.2% maximum decrease in total heat release, lower toxic CO yield, and formation of high-graphitized protective char layer. In addition, the mechanism for flame retardancy and toxicity suppression was proposed. It is reasonable to know that the improved flame-retardant performance for EP nanocomposites is attributed to tripartite cooperative effect from respective components (Co 2 P NPs and RGO) plus the heteroatom-doped carbon spheres.

Aminated multiwalled carbon nanotubes (A-MWCNT) were reacted with diphenylphosphinic chloride (DPP-Cl) to prepare the functionalized MWCNT (DPPA-MWCNT). A-MWCNT and DPPA-MWCNT were respectively mixed with polystyrene (PS) to obtain composites through the melt compounding method. SEM observations demonstrated that the DPPA-MWCNT nanofillers were more uniformly distributed within the PS matrix than A-MWCNT. PS/DPPA-MWCNT showed improved thermal stability, glass transition temperature, and tensile strength in comparison with PS/A-MWCNT, resulting from good dispersion and interfacial interactions between DPPA-MWCNT and PS matrix. The incorporation of DPPA-MWCNT to PS significantly reduced peak heat release rate, smoke production rate, and carbon monoxide and carbon dioxide release in cone calorimeter tests. The enhanced fire-retardant properties should be ascribed to the barrier effect of carbon nanotubes, which could provide enough time for DPPA-MWCNT and its functionalized groups to trap the degrading polymer radicals to catalyze char formation. The char layer served as an efficient insulating barrier to reduce the exposure of polymer matrix to an external heat source as well as retarding the flammable gases from feeding the flame.

De novo mutations (DNMs) are important in Autism Spectrum Disorder (ASD), but so far analyses have mainly been on the ~1.5% of the genome encoding genes. Here, we performed whole genome sequencing (WGS) of 200 ASD parent-child trios and characterized germline and somatic DNMs. We confirmed that the majority of germline DNMs (75.6%) originated from the father, and these increased significantly with paternal age only (p=4.2×10 -10 ). However, when clustered DNMs (those within 20kb) were found in ASD, not only did they mostly originate from the mother (p=7.7×10 -13 ), but they could also be found adjacent to de novo copy number variations (CNVs) where the mutation rate was significantly elevated (p=2.4×10 -24 ). By comparing DNMs detected in controls, we found a significant enrichment of predicted damaging DNMs in ASD cases (p=8.0×10 -9 ; OR=1.84), of which 15.6% (p=4.3×10 -3 ) and 22.5% (p=7.0×10 -5 ) were in the non-coding or genic non-coding, respectively. The non-coding elements most enriched for DNM were untranslated regions of genes, boundaries involved in exon-skipping and DNase I hypersensitive regions. Using microarrays and a novel outlier detection test, we also found aberrant methylation profiles in 2/185 (1.1%) of ASD cases. These same individuals carried independently identified DNMs in the ASD risk- and epigenetic- genes DNMT3A and ADNP. Our data begins to characterize different genome-wide DNMs, and highlight the contribution of non-coding variants, to the etiology of ASD.

A challenge in clinical genomics is to predict whether copy number variation (CNV) affecting a gene or multiple genes will manifest as disease. Increasing recognition of gene dosage effects in neurodevelopmental disorders prompted us to develop a computational approach based on critical-exon (highly expressed in brain, highly conserved) examination for potential etiologic effects. Using a large CNV dataset, our updated analyses revealed significant (P < 1.64 × 10(-15)) enrichment of critical-exons within rare CNVs in cases compared to controls. Separately, we used a weighted gene co-expression network analysis (WGCNA) to construct an unbiased protein module from prenatal and adult tissues and found it significantly enriched for critical exons in prenatal (P < 1.15 × 10(-50), OR = 2.11) and adult (P < 6.03 × 10(-18), OR = 1.55) tissues. WGCNA yielded 1,206 proteins for which we prioritized the corresponding genes as likely to have a role in neurodevelopmental disorders. We compared the gene lists obtained from critical-exon and WGCNA analysis and found 438 candidate genes associated with CNVs annotated as pathogenic, or as variants of uncertain significance (VOUS), from among 10,619 developmental delay cases. We identified genes containing CNVs previously considered to be VOUS to be new candidate genes for neurodevelopmental disorders (GIT1, MVB12B and PPP1R9A) demonstrating the utility of this strategy to index the clinical effects of CNVs.

Roifman Syndrome is a rare congenital disorder characterized by growth retardation, cognitive delay, spondyloepiphyseal dysplasia and antibody deficiency. Here we utilize whole-genome sequencing of Roifman Syndrome patients to reveal compound heterozygous rare variants that disrupt highly conserved positions of the RNU4ATAC small nuclear RNA gene, a minor spliceosome component that is essential for minor intron splicing. Targeted sequencing confirms allele segregation in six cases from four unrelated families. RNU4ATAC rare variants have been recently reported to cause microcephalic osteodysplastic primordial dwarfism, type I (MOPD1), whose phenotype is distinct from Roifman Syndrome. Strikingly, all six of the Roifman Syndrome cases have one variant that overlaps MOPD1-implicated structural elements, while the other variant overlaps a highly conserved structural element not previously implicated in disease. RNA-seq analysis confirms extensive and specific defects of minor intron splicing. Available allele frequency data suggest that recessive genetic disorders caused by RNU4ATAC rare variants may be more prevalent than previously reported.

Importance: The use of genome-wide tests to provide molecular diagnosis for individuals with autism spectrum disorder (ASD) requires more study., Objective: To perform chromosomal microarray analysis (CMA) and whole-exome sequencing (WES) in a heterogeneous group of children with ASD to determine the molecular diagnostic yield of these tests in a sample typical of a developmental pediatric clinic., Design, Setting, and Participants: The sample consisted of 258 consecutively ascertained unrelated children with ASD who underwent detailed assessments to define morphology scores based on the presence of major congenital abnormalities and minor physical anomalies. The children were recruited between 2008 and 2013 in Newfoundland and Labrador, Canada. The probands were stratified into 3 groups of increasing morphological severity: essential, equivocal, and complex (scores of 0-3, 4-5, and ≥6)., Exposures: All probands underwent CMA, with WES performed for 95 proband-parent trios., Main Outcomes and Measures: The overall molecular diagnostic yield for CMA and WES in a population-based ASD sample stratified in 3 phenotypic groups., Results: Of 258 probands, 24 (9.3%, 95%CI, 6.1%-13.5%) received a molecular diagnosis from CMA and 8 of 95 (8.4%, 95%CI, 3.7%-15.9%) from WES. The yields were statistically different between the morphological groups. Among the children who underwent both CMA and WES testing, the estimated proportion with an identifiable genetic etiology was 15.8% (95%CI, 9.1%-24.7%; 15/95 children). This included 2 children who received molecular diagnoses from both tests. The combined yield was significantly higher in the complex group when compared with the essential group (pairwise comparison, P = .002). [table: see text]., Conclusions and Relevance: Among a heterogeneous sample of children with ASD, the molecular diagnostic yields of CMA and WES were comparable, and the combined molecular diagnostic yield was higher in children with more complex morphological phenotypes in comparison with the children in the essential category. If replicated in additional populations, these findings may inform appropriate selection of molecular diagnostic testing for children affected by ASD.

Novel spinel copper cobaltate (CuCo2O4)/graphitic carbon nitride (g-C3N4) (named C-CuCo2O4) nanohybrids with different weight ratios of g-C3N4 to CuCo2O4 were successfully synthesized via a facile hydrothermal method. Then the nanohybrids were added into the thermoplastic polyurethane (TPU) matrix to prepare TPU nanocomposites using a master batch-melt compounding approach. Morphological analysis indicated that CuCo2O4 nanoparticles were uniformly distributed on g-C3N4 nanosheets. Thermal analysis revealed that C-CuCo2O4-7 (proportion of g-C3N4 to CuCo2O4 of 93/7) was an optimal nanohybrid for the properties improvement of TPU. Incorporation of C-CuCo2O4-7 into TPU led to significant improvements in the onset decomposition temperature, temperature at maximal mass loss rate and char yields. The heat release rate and total heat release of TPU/C-CuCo2O4-7 decreased by 37% and 31.3%, respectively, compared with those of pure TPU. Furthermore, the amounts of pyrolysis gaseous products, including combustible volatiles and carbon monoxide (CO), were remarkably reduced, whereas, non-flammable gas (carbon dioxide) increased. Excellent dispersion of C-CuCo2O4-7 in TPU host was achieved, due to the synergistic effect between g-C3N4 and CuCo2O4. Enhancements in the thermal stability and flame retardancy were attributed to the explanations that g-C3N4 nanosheets showed the physical barrier effect and catalytic nitrogen monoxide (NO) decomposition, and that CuCo2O4 catalyzes the reaction of CO with NO and increased char residues., (Copyright © 2015. Published by Elsevier B.V.)

Previously we showed that extravillous cytotrophoblast (EVT) outgrowth and migration on a collagen gel explant model were affected by exposure to decidual natural killer cells (dNK). This study investigates the molecular causes behind this phenomenon. Genome wide DNA methylation of exposed and unexposed EVT was assessed using the Illumina Infinium HumanMethylation450 BeadChip array (450 K array). We identified 444 differentially methylated CpG loci in dNK-treated EVT compared with medium control (P < 0.05). The genes associated with these loci had critical biological roles in cellular development, cellular growth and proliferation, cell signaling, cellular assembly and organization by Ingenuity Pathway Analysis (IPA). Furthermore, 23 mobility-related genes were identified by IPA from dNK-treated EVT. Among these genes, CLDN4 (encoding claudin-4) and FUT4 (encoding fucosyltransferase IV) were chosen for follow-up studies because of their biological relevance from research on tumor cells. The results showed that the mRNA and protein expressions of both CLDN4 and FUT4 in dNK-treated EVT were significantly reduced compared with control (P < 0.01 for both CLDN4 and FUT4 mRNA expression; P < 0.001 for CLDN4 and P < 0.01 for FUT4 protein expression), and were inversely correlated with DNA methylation. Knocking down CLDN4 and FUT4 by small interfering RNA reduced trophoblast invasion, possibly through the altered matrix metalloproteinase (MMP)-2 and/or MMP-9 expression and activity. Taken together, dNK alter EVT mobility at least partially in association with an alteration of DNA methylation profile. Hypermethylation of CLDN4 and FUT4 reduces protein expression. CLDN4 and FUT4 are representative genes that participate in modulating trophoblast mobility., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Autism spectrum disorder (ASD) is genetically heterogeneous, with evidence for hundreds of susceptibility loci. Previous microarray and exome-sequencing studies have examined portions of the genome in simplex families (parents and one ASD-affected child) having presumed sporadic forms of the disorder. We used whole-genome sequencing (WGS) of 85 quartet families (parents and two ASD-affected siblings), consisting of 170 individuals with ASD, to generate a comprehensive data resource encompassing all classes of genetic variation (including noncoding variants) and accompanying phenotypes, in apparently familial forms of ASD. By examining de novo and rare inherited single-nucleotide and structural variations in genes previously reported to be associated with ASD or other neurodevelopmental disorders, we found that some (69.4%) of the affected siblings carried different ASD-relevant mutations. These siblings with discordant mutations tended to demonstrate more clinical variability than those who shared a risk variant. Our study emphasizes that substantial genetic heterogeneity exists in ASD, necessitating the use of WGS to delineate all genic and non-genic susceptibility variants in research and in clinical diagnostics.

Copy number variation has emerged as an important cause of phenotypic variation, particularly in relation to some complex disorders. Autism spectrum disorder (ASD) is one such disorder, in which evidence is emerging for an etiological role for some rare penetrant de novo and rare inherited copy number variants (CNVs). De novo variation, however, does not always explain the familial nature of ASD, leaving a gap in our knowledge concerning the heritable genetic causes of this disorder. Extended pedigrees, in which several members have ASD, provide an opportunity to investigate inherited genetic risk factors. In this current study, we recruited 19 extended ASD pedigrees, and, using the Illumina HumanOmni2.5 BeadChip, conducted genome-wide CNV interrogation. We found no definitive evidence of an etiological role for segregating CNVs in these pedigrees, and no evidence that linkage signals in these pedigrees are explained by segregating CNVs. However, a small number of putative de novo variants were transmitted from BAP parents to their ASD offspring, and evidence emerged for a rare duplication CNV at 11p13.3 harboring two putative 'developmental/neuropsychiatric' susceptibility gene(s), GSTP1 and NDUFV1.

To facilitate precision medicine and whole-genome annotation, we developed a machine-learning technique that scores how strongly genetic variants affect RNA splicing, whose alteration contributes to many diseases. Analysis of more than 650,000 intronic and exonic variants revealed widespread patterns of mutation-driven aberrant splicing. Intronic disease mutations that are more than 30 nucleotides from any splice site alter splicing nine times as often as common variants, and missense exonic disease mutations that have the least impact on protein function are five times as likely as others to alter splicing. We detected tens of thousands of disease-causing mutations, including those involved in cancers and spinal muscular atrophy. Examination of intronic and exonic variants found using whole-genome sequencing of individuals with autism revealed misspliced genes with neurodevelopmental phenotypes. Our approach provides evidence for causal variants and should enable new discoveries in precision medicine., (Copyright © 2015, American Association for the Advancement of Science.)

The genetic architecture of autism spectrum disorder involves the interplay of common and rare variants and their impact on hundreds of genes. Using exome sequencing, here we show that analysis of rare coding variation in 3,871 autism cases and 9,937 ancestry-matched or parental controls implicates 22 autosomal genes at a false discovery rate (FDR) < 0.05, plus a set of 107 autosomal genes strongly enriched for those likely to affect risk (FDR < 0.30). These 107 genes, which show unusual evolutionary constraint against mutations, incur de novo loss-of-function mutations in over 5% of autistic subjects. Many of the genes implicated encode proteins for synaptic formation, transcriptional regulation and chromatin-remodelling pathways. These include voltage-gated ion channels regulating the propagation of action potentials, pacemaking and excitability-transcription coupling, as well as histone-modifying enzymes and chromatin remodellers-most prominently those that mediate post-translational lysine methylation/demethylation modifications of histones.

A universal challenge in genetic studies of autism spectrum disorders (ASDs) is determining whether a given DNA sequence alteration will manifest as disease. Among different population controls, we observed, for specific exons, an inverse correlation between exon expression level in brain and burden of rare missense mutations. For genes that harbor de novo mutations predicted to be deleterious, we found that specific critical exons were significantly enriched in individuals with ASD relative to their siblings without ASD (P < 1.13 × 10(-38); odds ratio (OR) = 2.40). Furthermore, our analysis of genes with high exonic expression in brain and low burden of rare mutations demonstrated enrichment for known ASD-associated genes (P < 3.40 × 10(-11); OR = 6.08) and ASD-relevant fragile-X protein targets (P < 2.91 × 10(-157); OR = 9.52). Our results suggest that brain-expressed exons under purifying selection should be prioritized in genotype-phenotype studies for ASD and related neurodevelopmental conditions.

Rare copy number variants (CNVs) disrupting ASTN2 or both ASTN2 and TRIM32 have been reported at 9q33.1 by genome-wide studies in a few individuals with neurodevelopmental disorders (NDDs). The vertebrate-specific astrotactins, ASTN2 and its paralog ASTN1, have key roles in glial-guided neuronal migration during brain development. To determine the prevalence of astrotactin mutations and delineate their associated phenotypic spectrum, we screened ASTN2/TRIM32 and ASTN1 (1q25.2) for exonic CNVs in clinical microarray data from 89 985 individuals across 10 sites, including 64 114 NDD subjects. In this clinical dataset, we identified 46 deletions and 12 duplications affecting ASTN2. Deletions of ASTN1 were much rarer. Deletions near the 3' terminus of ASTN2, which would disrupt all transcript isoforms (a subset of these deletions also included TRIM32), were significantly enriched in the NDD subjects (P = 0.002) compared with 44 085 population-based controls. Frequent phenotypes observed in individuals with such deletions include autism spectrum disorder (ASD), attention deficit hyperactivity disorder (ADHD), speech delay, anxiety and obsessive compulsive disorder (OCD). The 3'-terminal ASTN2 deletions were significantly enriched compared with controls in males with NDDs, but not in females. Upon quantifying ASTN2 human brain RNA, we observed shorter isoforms expressed from an alternative transcription start site of recent evolutionary origin near the 3' end. Spatiotemporal expression profiling in the human brain revealed consistently high ASTN1 expression while ASTN2 expression peaked in the early embryonic neocortex and postnatal cerebellar cortex. Our findings shed new light on the role of the astrotactins in psychopathology and their interplay in human neurodevelopment.

Objective: To develop a novel clinical test using microarray technology as a high-resolution alternative to current methods for detection of known and novel microdeletions on the Y chromosome., Design: Custom Agilent 8x15K array comparative genomic hybridization (aCGH) with 10,162 probes on an average probe spacing of 2.5 kb across the euchromatic region of the Y chromosome., Setting: Clinical diagnostic laboratory., Patient(s): Men with infertility (n = 104) and controls with proven fertility (n = 148)., Intervention(s): Microarray genotyping of DNA., Main Outcome Measure(s): Gene copy number variation determined by log ratio of probe signal intensity against a DNA reference., Result(s): Our aCGH experiments found all known AZF microdeletions as well as additional unbalanced structural alterations. In addition to complete AZF microdeletions, we found that AZFc partial deletions represent a risk factor for male infertility. In total, aCGH-based detection achieved a diagnostic yield of ∼11% and also revealed additional potentially etiologic copy number variations requiring further characterization., Conclusion(s): The aCGH approach is a reliable high-resolution alternative to multiplex polymerase chain reaction for the discovery of pathogenic chromosome Y microdeletions in male infertility., (Copyright © 2014 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

We observed that current high-throughput sequencing approaches only detected a fraction of the full size-spectrum of insertions, deletions, and copy number variants compared with a previously published, Sanger-sequenced human genome. The sensitivity for detection was the lowest in the 100- to 10,000-bp size range, and at DNA repeats, with copy number gains harder to delineate than losses. We discuss strategies for discovering the full spectrum of genetic variation necessary for disease association studies.

A series of sodium alginate (SA) nanocomposite films with different loading levels of graphitic-like carbon nitride (g-C3N4) were fabricated via the casting technique. The structure and morphology of nanocomposite films were investigated by X-ray powder diffraction, Fourier transform infrared spectroscopy, scanning electron microscopy, and transmission electron microscopy. Thermogravimetric analysis results suggested that thermal stability of all the nanocomposite films was enhanced significantly, including initial thermal degradation temperature increased by 29.1 °C and half thermal degradation temperature improved by 118.2 °C. Mechanical properties characterized by tensile testing and dynamic mechanical analysis measurements were also reinforced remarkably. With addition of 6.0 wt % g-C3N4, the tensile strength of SA nanocomposite films was dramatically enhanced by 103%, while the Young's modulus remarkably increased from 60 to 3540 MPa. Moreover, the storage modulus significantly improved by 34.5% was observed at loadings as low as 2.0 wt %. These enhancements were further investigated by means of differential scanning calorimetry and real time Fourier transform infrared spectra. A new perspective of balance was proposed to explain the improvement of those properties for the first time. At lower than 1.0 wt % loading, most of the g-C3N4 nanosheets were discrete in the SA matrix, resulting in improved thermal stability and mechanical properties; above 1.0 wt % and below 6.0 wt % content, the aggregation was present in SA host coupled with insufficient hydrogen bondings limiting the barrier for heat and leading to the earlier degradation and poor dispersion; at 6.0 wt % addition, the favorable balance was established with enhanced thermal and mechanical performances. However, the balance point of 2.0 wt % from dynamic mechanical analysis was due to combination of temperature and agglomeration. The work may contribute to a potential research approach for other nanocomposites.

Over the past decade, the Database of Genomic Variants (DGV; http://dgv.tcag.ca/) has provided a publicly accessible, comprehensive curated catalogue of structural variation (SV) found in the genomes of control individuals from worldwide populations. Here, we describe updates and new features, which have expanded the utility of DGV for both the basic research and clinical diagnostic communities. The current version of DGV consists of 55 published studies, comprising >2.5 million entries identified in >22,300 genomes. Studies included in DGV are selected from the accessioned data sets in the archival SV databases dbVar (NCBI) and DGVa (EBI), and then further curated for accuracy and validity. The core visualization tool (gbrowse) has been upgraded with additional functions to facilitate data analysis and comparison, and a new query tool has been developed to provide flexible and interactive access to the data. The content from DGV is regularly incorporated into other large-scale genome reference databases and represents a standard data resource for new product and database development, in particular for copy number variation testing in clinical labs. The accurate cataloguing of variants in DGV will continue to enable medical genetics and genome sequencing research.

Pre-eclampsia is a serious complication of pregnancy that can affect both maternal and fetal outcomes. Early-onset pre-eclampsia (EOPET) is a severe form of pre-eclampsia that is associated with altered physiological characteristics and gene expression in the placenta. DNA methylation is a relatively stable epigenetic modification to DNA that can reflect gene expression, and can provide insight into the mechanisms underlying such expression changes. This case-control study focused on DNA methylation and gene expression of whole chorionic villi samples from 20 EOPET placentas and 20 gestational age-matched controls from pre-term births. DNA methylation was also assessed in placentas affected by late-onset pre-eclampsia (LOPET) and normotensive intrauterine growth restriction (nIUGR). The Illumina HumanMethylation450 BeadChip was used to assess DNA methylation at >480 000 cytosine-guanine dinucleotide (CpG) sites. The Illumina HT-12v4 Expression BeadChip was used to assess gene expression of >45 000 transcripts in a subset of cases and controls. DNA methylation analysis by pyrosequencing was used to follow-up the initial findings in four genes with a larger cohort of cases and controls, including nIUGR and LOPET placentas. Bioinformatic analysis was used to identify overrepresentation of gene ontology categories and transcription factor binding motifs. We identified 38 840 CpG sites with significant (false discovery rate <0.01) DNA methylation alterations in EOPET, of which 282 had >12.5% methylation difference compared with the controls. Significant sites were enriched at the enhancers and low CpG density regions of the associated genes and the majority (74.5%) of these sites were hypomethylated in EOPET. EOPET, but not associated clinical features, such as intrauterine growth restriction (IUGR), presented a distinct DNA methylation profile. CpG sites from four genes relevant to pre-eclampsia (INHBA, BHLHE40, SLC2A1 and ADAM12) showed different extent of changes in LOPET and nIUGR. Genome-wide expression in a subset of samples showed that some of the gene expression changes were negatively correlated with DNA methylation changes, particularly for genes that are responsible for angiogenesis (such as EPAS1 and FLT1). Results could be confounded by altered cell populations in abnormal placentas. Larger sample sizes are needed to fully address the possibility of sub-profiles of methylation within the EOPET cohort. Based on DNA methylation profiling, we conclude that there are widespread DNA methylation alterations in EOPET that may be associated with changes in placental function. This property may provide a useful tool for early screening of such placentas. This study identifies DNA methylation changes at many loci previously reported to have altered gene expression in EOPET placentas, as well as in novel biologically relevant genes we confirmed to be differentially expressed. These results may be useful for DNA- methylation-based non-invasive prenatal diagnosis of at-risk pregnancies.

Autism Spectrum Disorder (ASD) demonstrates high heritability and familial clustering, yet the genetic causes remain only partially understood as a result of extensive clinical and genomic heterogeneity. Whole-genome sequencing (WGS) shows promise as a tool for identifying ASD risk genes as well as unreported mutations in known loci, but an assessment of its full utility in an ASD group has not been performed. We used WGS to examine 32 families with ASD to detect de novo or rare inherited genetic variants predicted to be deleterious (loss-of-function and damaging missense mutations). Among ASD probands, we identified deleterious de novo mutations in six of 32 (19%) families and X-linked or autosomal inherited alterations in ten of 32 (31%) families (some had combinations of mutations). The proportion of families identified with such putative mutations was larger than has been previously reported; this yield was in part due to the comprehensive and uniform coverage afforded by WGS. Deleterious variants were found in four unrecognized, nine known, and eight candidate ASD risk genes. Examples include CAPRIN1 and AFF2 (both linked to FMR1, which is involved in fragile X syndrome), VIP (involved in social-cognitive deficits), and other genes such as SCN2A and KCNQ2 (linked to epilepsy), NRXN1, and CHD7, which causes ASD-associated CHARGE syndrome. Taken together, these results suggest that WGS and thorough bioinformatic analyses for de novo and rare inherited mutations will improve the detection of genetic variants likely to be associated with ASD or its accompanying clinical symptoms., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

The mechanisms by which the placenta adapts to exogenous stimuli to create a stable and healthy environment for the growing fetus are not well known. Low oxygen tension influences placental function, and is associated with preeclampsia, a condition displaying altered development of placental trophoblast. We hypothesized that oxygen tension affects villous trophoblast by modulation of gene expression through DNA methylation. We used the Infinium HumanMethylation450 BeadChip array to compare the DNA methylation profile of primary cultures of human cytotrophoblasts and syncytiotrophoblasts under < 1%, 8% and 20% oxygen levels. We found no effect of oxygen tension on average DNA methylation for either cell phenotype, but a set of loci became hypermethylated in cytotrophoblasts exposed for 24 h to < 1% oxygen, as compared with those exposed to 8% or 20% oxygen. Hypermethylation with low oxygen tension was independently confirmed by bisulfite-pyrosequencing in a subset of functionally relevant genes including CD59, CFB, GRAM3 and ZNF217. Intriguingly, 70 out of the 147 CpGs that became hypermethylated in < 1% oxygen overlapped with CpG sites that became hypomethylated upon differentiation of cytotrophoblasts into syncytiotrophoblasts. Furthermore, the preponderance of altered sites was located at AP-1 binding sites. We suggest that AP-1 expression is triggered by hypoxia and interacts with DNA methyltransferases (DNMTs) to target methylation at specific sites in the genome, thus causing suppression of the associated genes that are responsible for differentiation of villous cytotrophoblast to syncytiotrophoblast.

Background: The human placenta facilitates the exchange of nutrients, gas and waste between the fetal and maternal circulations. It also protects the fetus from the maternal immune response. Due to its role at the feto-maternal interface, the placenta is subject to many environmental exposures that can potentially alter its epigenetic profile. Previous studies have reported gene expression differences in placenta over gestation, as well as inter-individual variation in expression of some genes. However, the factors contributing to this variation in gene expression remain poorly understood., Results: In this study, we performed a genome-wide DNA methylation analysis of gene promoters in placenta tissue from three pregnancy trimesters. We identified large-scale differences in DNA methylation levels between first, second and third trimesters, with an overall progressive increase in average methylation from first to third trimester. The most differentially methylated genes included many immune regulators, reflecting the change in placental immuno-modulation as pregnancy progresses. We also detected increased inter-individual variation in the third trimester relative to first and second, supporting an accumulation of environmentally induced (or stochastic) changes in DNA methylation pattern. These highly variable genes were enriched for those involved in amino acid and other metabolic pathways, potentially reflecting the adaptation of the human placenta to different environments., Conclusions: The identification of cellular pathways subject to drift in response to environmental influences provide a basis for future studies examining the role of specific environmental factors on DNA methylation pattern and placenta-associated adverse pregnancy outcomes.

Background: Genomic imprinting is an important epigenetic process involved in regulating placental and foetal growth. Imprinted genes are typically associated with differentially methylated regions (DMRs) whereby one of the two alleles is DNA methylated depending on the parent of origin. Identifying imprinted DMRs in humans is complicated by species- and tissue-specific differences in imprinting status and the presence of multiple regulatory regions associated with a particular gene, only some of which may be imprinted. In this study, we have taken advantage of the unbalanced parental genomic constitutions in triploidies to further characterize human DMRs associated with known imprinted genes and identify novel imprinted DMRs., Results: By comparing the promoter methylation status of over 14,000 genes in human placentas from ten diandries (extra paternal haploid set) and ten digynies (extra maternal haploid set) and using 6 complete hydatidiform moles (paternal origin) and ten chromosomally normal placentas for comparison, we identified 62 genes with apparently imprinted DMRs (false discovery rate <0.1%). Of these 62 genes, 11 have been reported previously as DMRs that act as imprinting control regions, and the observed parental methylation patterns were concordant with those previously reported. We demonstrated that novel imprinted genes, such as FAM50B, as well as novel imprinted DMRs associated with known imprinted genes (for example, CDKN1C and RASGRF1) can be identified by using this approach. Furthermore, we have demonstrated how comparison of DNA methylation for known imprinted genes (for example, GNAS and CDKN1C) between placentas of different gestations and other somatic tissues (brain, kidney, muscle and blood) provides a detailed analysis of specific CpG sites associated with tissue-specific imprinting and gestational age-specific methylation., Conclusions: DNA methylation profiling of triploidies in different tissues and developmental ages can be a powerful and effective way to map and characterize imprinted regions in the genome.

Background: Development of human tissue is influenced by a combination of intrinsic biological signals and extrinsic environmental stimuli, both of which are mediated by epigenetic regulation, including DNA methylation. However, little is currently known of the normal acquisition or loss of epigenetic markers during fetal and postnatal development., Results: The DNA methylation status of over 1000 CpGs located in the regulatory regions of nearly 800 genes was evaluated in five somatic tissues (brain, kidney, lung, muscle and skin) from eight normal second-trimester fetuses. Tissue-specific differentially methylated regions (tDMRs) were identified in 195 such loci. However, comparison with corresponding data from trisomic fetuses (five trisomy 21 and four trisomy 18) revealed relatively few DNA methylation differences associated with trisomy, despite such conditions having a profound effect on development. Of interest, only 17% of the identified fetal tDMRs were found to maintain this same tissue-specific DNA methylation in adult tissues. Furthermore, 10% of the sites analyzed, including sites associated with imprinted genes, had a DNA methylation difference of >40% between fetus and adult. This plasticity of DNA methylation over development was further confirmed by comparison with similar data from embryonic stem cells, with the most altered methylation levels being linked to domains with bivalent histone modifications., Conclusions: Most fetal tDMRs seem to reflect transient DNA methylation changes during development rather than permanent epigenetic signatures. The extensive tissue-specific and developmental-stage specific nature of DNA methylation will need to be elucidated to identify abnormal patterns of DNA methylation associated with abnormal development or disease.

Genetic and epigenetic studies of the human placenta can help to clarify the underlying mechanisms of placenta-associated diseases. However, such studies have also revealed a considerable degree of within- and between-placenta variability, which can be attributed to a variety of influences. We illustrate the inherent heterogeneity in the placenta using examples from two types of studies: 1) chromosomal mosaicism and 2) DNA methylation variation. We discuss the factors that may influence the distribution of variation and how, understanding the source of this variation is important for interpreting data used to investigate and predict clinical outcomes., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

An imbalance of imprinted gene expression within 11p15.5 is observed in Beckwith-Wiedemann syndrome (BWS), as well as in a variety of placental abnormalities including complete hydatidiform mole (CHM), placental mesenchymal dysplasia (PMD) and triploidy. To facilitate the diagnosis of epigenetic errors and chromosomal imbalance of 11p15.5, we validated a pyrosequencing assay to measure methylation at KvDMR1 using blood samples from 13 BWS cases, 8 of which showed reduced methylation as compared to control blood. An imbalance between maternal and paternal genomes as is found in triploidy, CHM or PMD was also associated with altered KvDMR1 methylation. A reciprocal pattern of methylation was obtained in the triploid cases by assaying the proximal 11p15.5 ICR associated with H19. To distinguish chromosome 11 specific alterations from whole genome imbalance, other imprinted differentially methylated regions (DMRs) can be utilized. Thus, pyrosequencing assays for DMRs associated with SGCE, SNRPN, and MEST were also compared for their utility in diagnosing parental imbalance in placental samples. While each of these assays could successfully distinguish parental origin of triploidy, SGCE showed the clearest separation between groups. The combined use of a chromosome 11p15.5 assay (e.g. KvDMR1 or H19-ICR) and non-chromosome 11 assay (e.g. SGCE) provides a potentially valuable diagnostic tool in the rapid screening of methylation errors in placental disorders. These results also show the maintenance of imprinting status at these loci in the human placenta, even in the presence of abnormal pathology., (© 2010 John Wiley & Sons A/S.)

Unlabelled: Obtaining representative samples from a term placenta for gene-expression studies is confounded by both within placental heterogeneity and sampling effects such as sample location and processing time. Epigenetic processes involved in the regulation of gene expression, such as DNA methylation, may show similar variability, but are less well studied. Therefore, we investigated the nature of within and between- placenta variation in gene expression and DNA methylation of genes that were chosen for being differentially expressed or methylated by cell type within the placenta., Methods: In total, two or more samples from each of 38 normal term placentae were utilized. The expression levels of CDH1, CDH11, ID2, PLAC1 and KISS1 were evaluated by real-time PCR. DNA methylation levels of LINE1 elements and CpGs within the promoter regions of KISS1, PTPN6, CASP8, and APC were similarly quantified by pyrosequencing., Results: Despite considerable sample-to-sample variability within each placenta, the within-placenta correlation for both gene expression and methylation was significant for each studied gene. Most of this variability was not due to sample location. However, between placental differences in gene expression were inflated by the dramatic effect of processing time (0-24 h) on mRNA levels, particularly for PLAC1 and KISS1 (both expressed in the apical syncytiotrophoblast). In contrast, DNA methylation levels remained relatively constant over this same time period., Conclusion: Due to extensive site-to-site variability, multiple sampled sites are needed to accurately represent a placenta for molecular studies. Furthermore, mRNA quantitation of some genes may be hampered by its rapid degradation post-delivery (and possibly perinatally) and thus processing time should be considered in such analyses. Within-placenta correlations in expression and methylation from unrelated genes raise the possibility that methylation and expression variation may potentially reflect cell composition differences between samples rather than true differences occurring at the cellular level., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregnancy outcomes, but their underlying causes are mostly unknown. Although multiple studies have investigated gene expression changes in these disorders, few studies have examined epigenetic changes. Analysis of the DNA methylation pattern associated with such pregnancies provides an alternative approach to identifying cellular changes involved in these disorders. We analyzed methylation of 1505 CpG sites associated with 807 genes in 26 placentas from early-onset preeclampsia (EOPET), late-onset preeclampsia, IUGR and control subjects using an Illumina GoldenGate Methylation panel. Thirty-four loci were hypomethylated (false discovery rate <10% and methylation difference >10%) in the early-onset preeclamptic placentas while no and only five differentially methylated loci were found in late-onset preeclamptic and IUGR placentas, respectively. Hypomethylation of 4 loci in EOPET was further confirmed by bisulfite pyrosequencing of 26 independent placental samples. The promoter of TIMP3 was confirmed to be significantly hypomethylated in EOPET placentas (P=0.00001). Our results suggest that gene-specific hypomethylation may be a common phenomenon in EOPET placentas, and that TIMP3 could serve as a potential prenatal diagnostic marker for EOPET.

Interindividual variation in DNA-methylation level is widespread in the human genome, despite its critical role in regulating gene expression. The nature of this variation, including its tissue-specific nature, and the role it may play in human phenotypic variation and disease is still poorly characterized. The placenta plays a critical role in regulating fetal growth and development in ways that have lifelong effects on health. To identify genes with a high degree of interindividual DNA methylation variation in the human placenta, we surveyed the human genome using the Illumina GoldenGate Methylation Cancer panel targeting 1505 CpG sites of 807 genes. While many sites show a continuous pattern of methylation levels, WNT2, TUSC3 and EPHB4 were identified to have a polymorphic "on-or-off" pattern of DNA methylation variation at their promoter region which was confirmed by pyrosequencing. Methylation of these genes can be found in 7%-25% of over 100 placentas tested. The methylation state at the promoter of these genes is concordant with mRNA allelic expression. In three informative cases TUSC3 was observed to be methylated on the maternal allele, and it is thus possible this represents a polymorphically imprinted gene. Furthermore, TUSC3 promoter methylation showed evidence for association with preeclampsia. A biological significance of these methylation allelic polymorphisms (MAPs) to human placental diversity is further implied by their placental specificity and absence in mouse. An extended study of blood suggests that MAPs may also be found in other tissues, implicating their utility for tissue-specific association with complex disorders. The identification of such "epipolymorphism" in other tissues and their use in association studies, should improve our understanding of interindividual phenotypic variability and complex disease susceptibility.

The pseudomalignant nature of the placenta prompted us to search for tumor suppressor gene hypermethylation, a phenomenon widely reported in cancer, in the human placenta. Nine tumor suppressor genes were studied. Hypermethylation of the Ras association domain family 1 A (RASSF1A) gene was found in human placentas from all three trimesters of pregnancy but was absent in other fetal tissues. Hypermethylation of Rassf1 was similarly observed in placentas from the rhesus monkey but not the mouse. An inverse relationship between RASSF1A promoter methylation and gene expression was demonstrated by bisulfite sequencing of microdissected placental cells and immunohistochemical staining of placental tissue sections using an anti-RASSF1A antibody. Treatment of choriocarcinoma cell lines, JAR and JEG3, by 5-aza-2'-deoxycytidine and trichostatin A led to reduction in RASSF1A methylation but increased expression. These observations extend the analogy between the primate placenta and malignant tumors to the epigenetic level.

A hybrid neural network model, based on the fusion of fuzzy adaptive resonance theory (FA ART) and the general regression neural network (GRNN), is proposed in this paper. Both FA and the GRNN are incremental learning systems and are very fast in network training. The proposed hybrid model, denoted as GRNNFA, is able to retain these advantages and, at the same time, to reduce the computational requirements in calculating and storing information of the kernels. A clustering version of the GRNN is designed with data compression by FA for noise removal. An adaptive gradient-based kernel width optimization algorithm has also been devised. Convergence of the gradient descent algorithm can be accelerated by the geometric incremental growth of the updating factor. A series of experiments with four benchmark datasets have been conducted to assess and compare effectiveness of GRNNFA with other approaches. The GRNNFA model is also employed in a novel application task for predicting the evacuation time of patrons at typical karaoke centers in Hong Kong in the event of fire. The results positively demonstrate the applicability of GRNNFA in noisy data regression problems.