Prevalence and Clinical Features of Inflammatory Bowel Diseases Associated With Monogenic Variants, Identified by Whole-Exome Sequencing in 1000 Children at a Single Center.

BACKGROUND & AIMS: A proportion of infants and young children with inflammatory bowel diseases (IBDs) have subtypes associated with a single gene variant (monogenic IBD). We aimed to determine the prevalence of monogenic disease in a cohort of pediatric patients with IBD. METHODS: We performed whole-exome sequencing analyses of blood samples from an unselected cohort of 1005 children with IBD, aged 0-18 years (median age at diagnosis, 11.96 years) at a single center in Canada and their family members (2305 samples total). Variants believed to cause IBD were validated using Sanger sequencing. Biopsies from patients were analyzed by immunofluorescence and histochemical analyses. RESULTS: We identified 40 rare variants associated with 21 monogenic genes among 31 of the 1005 children with IBD (including 5 variants in XIAP, 3 in DOCK8, and 2 each in FOXP3, GUCY2C, and LRBA). These variants occurred in 7.8% of children younger than 6 years and 2.3% of children aged 6-18 years. Of the 17 patients with monogenic Crohn's disease, 35% had abdominal pain, 24% had nonbloody loose stool, 18% had vomiting, 18% had weight loss, and 5% had intermittent bloody loose stool. The 14 patients with monogenic ulcerative colitis or IBD-unclassified received their diagnosis at a younger age, and their most predominant feature was bloody loose stool (78%). Features associated with monogenic IBD, compared to cases of IBD not associated with a single variant, were age of onset younger than 2 years (odds ratio [OR], 6.30; P = .020), family history of autoimmune disease (OR, 5.12; P = .002), extra-intestinal manifestations (OR, 15.36; P < .0001), and surgery (OR, 3.42; P = .042). Seventeen patients had variants in genes that could be corrected with allogeneic hematopoietic stem cell transplantation. CONCLUSIONS: In whole-exome sequencing analyses of more than 1000 children with IBD at a single center, we found that 3% had rare variants in genes previously associated with pediatric IBD. These were associated with different IBD phenotypes, and 1% of the patients had variants that could be potentially corrected with allogeneic hematopoietic stem cell transplantation. Monogenic IBD is rare, but should be considered in analysis of all patients with pediatric onset of IBD.

Early Procedural Pain Is Associated with Regionally-Specific Alterations in Thalamic Development in Preterm Neonates.

Very preterm human neonates are exposed to numerous invasive procedures as part of life-saving care. Evidence suggests that repetitive neonatal procedural pain precedes long-term alterations in brain development. However, to date the link between pain and brain development has limited temporal and anatomic specificity. We hypothesized that early exposure to painful stimuli during a period of rapid brain development, before pain modulatory systems reach maturity, will predict pronounced changes in thalamic development, and thereby cognitive and motor function. In a prospective cohort study, 155 very preterm neonates (82 males, 73 females) born 24-32 weeks' gestation underwent two MRIs at median postmenstrual ages 32 and 40 weeks that included structural, metabolic, and diffusion imaging. Detailed day-by-day clinical data were collected. Cognitive and motor abilities were assessed at 3 years, corrected age. The association of early (skin breaks, birth-Scan 1) and late pain (skin breaks, Scans 1-2) with thalamic volumes and N-acetylaspartate (NAA)/choline (Cho), and fractional anisotropy of white-matter pathways was assessed. Early pain was associated with slower thalamic macrostructural growth, most pronounced in extremely premature neonates. Deformation-based morphometry analyses confirmed early pain-related volume losses were localized to somatosensory regions. In extremely preterm neonates early pain was associated with decreased thalamic NAA/Cho and microstructural alterations in thalamocortical pathways. Thalamic growth was in turn related to cognitive and motor outcomes. We observed regionally-specific alterations in the lateral thalamus and thalamocortical pathways in extremely preterm neonates exposed to more procedural pain. Findings suggest a sensitive period leading to lasting alterations in somatosensory-system development.SIGNIFICANCE STATEMENT Early exposure to repetitive procedural pain in very preterm neonates may disrupt the development of regions involved in somatosensory processing, leading to poor functional outcomes. We demonstrate that early pain is associated with thalamic volume loss in the territory of the somatosensory thalamus and is accompanied by disruptions thalamic metabolic growth and thalamocortical pathway maturation, particularly in extremely preterm neonates. Thalamic growth was associated with cognitive and motor outcome at 3 years corrected age. Findings provide evidence for a developmentally sensitive period whereby subcortical structures in young neonates may be most vulnerable to procedural pain. Furthermore, results suggest that the thalamus may play a key role underlying the association between neonatal pain and poor neurodevelopmental outcomes in these high-risk neonates.

Multiple Postnatal Infections in Newborns Born Preterm Predict Delayed Maturation of Motor Pathways at Term-Equivalent Age with Poorer Motor Outcomes at 3 Years.

OBJECTIVES: To evaluate whether the number of postnatal infections is associated with abnormal white matter maturation and poorer motor neurodevelopmental outcomes at 36 months of corrected age. STUDY DESIGN: A prospective longitudinal cohort study was undertaken of 219 newborns born preterm at 24-32 weeks of gestational age recruited between 2006 and 2013 with magnetic resonance imaging of the brain both early in life and at term-equivalent age. Postnatal infection was defined as any clinical infection or positive culture ≥72 hours after birth. White matter maturation was assessed by magnetic resonance spectroscopic imaging, magnetic resonance diffusion tensor imaging, and tract-based spatial statistics. Neurodevelopmental outcomes were assessed in 175 (82% of survivors) infants with Bayley Scales of Infant and Toddler Development-III composite scores and Peabody Developmental Motor Scales at 35 months of corrected age (IQR 34-37 months). Infection groups were compared via the Fisher exact test, Kruskal-Wallis test, and generalized estimating equations. RESULTS: Of 219 neonates born preterm (median gestational age 27.9 weeks), 109 (50%) had no postnatal infection, 83 (38%) had 1 or 2 infections, and 27 (12%) had ≥3 infections. Infants with postnatal infections had more cerebellar hemorrhage. Infants with ≥3 infections had lower N-acetylaspartate/choline in the white matter and basal ganglia regions, lower fractional anisotropy in the posterior limb of the internal capsule, and poorer maturation of the corpus callosum, optic radiations, and posterior limb of the internal capsule on tract-based spatial statistics analysis as well as poorer Bayley Scales of Infant and Toddler Development-III (P = .02) and Peabody Developmental Motor Scales, Second Edition, motor scores (P < .01). CONCLUSIONS: In newborns born preterm, ≥3 postnatal infections predict impaired development of the motor pathways and poorer motor outcomes in early childhood.

An MRspec database query and visualization engine with applications as a clinical diagnostic and research tool.

PURPOSE: Proton magnetic resonance spectroscopy (MRspec), one of the very few techniques for in vivo assessment of neuro-metabolic profiles, is often complicated by lack of standard population norms and paucity of computational tools. METHODS: 7035 scans and clinical information from 4430 pediatric patients were collected from 2008 to 2014. Scans were conducted using a 1.5T (n=3664) or 3T scanner (n=3371), and with either a long (144ms, n=5559) or short echo time (35ms, n=1476). 3055 of these scans were localized in the basal ganglia (BG), 1211 in parieto-occipital white matter (WM). 34 metabolites were quantified using LCModel. A web application using MySQL, Python and Flask was developed to facilitate the exploration of the data set. RESULTS: Already piloting the application revealed numerous insights. (1), N-acetylaspartate (NAA) increased throughout all ages. During early infancy, total choline was highly varied and myo-inositol demonstrated a downward trend. (2), Total creatine (tCr) and creatine increased throughout childhood and adolescence, though phosphocreatine (PCr) remained constant beyond 200days. (3), tCr was higher in BG than WM. (4), No obvious gender-related differences were observed. (5), Field strength affects quantification using LCModel for some metabolites, most prominently for tCr and total NAA. (6), Outlier analysis identified patients treated with vigabatrin through elevated γ-aminobutyrate, and patients with Klippel-Feil syndrome, Leigh disease and L2-hydroxyglutaric aciduria through low choline in BG. CONCLUSIONS: We have established the largest MRSpec database and developed a robust and flexible computational tool for facilitating the exploration of vast metabolite datasets that proved its value for discovering neurochemical trends for clinical diagnosis, treatment monitoring, and research. Open access will lead to its widespread use, improving the diagnostic yield and contributing to better understanding of metabolic processes and conditions in the brain.

Early postnatal docosahexaenoic acid levels and improved preterm brain development.

BACKGROUND: Preterm birth has a dramatic impact on polyunsaturated fatty acid exposures for the developing brain. This study examined the association between postnatal fatty acid levels and measures of brain injury and development, as well as outcomes. METHODS: A cohort of 60 preterm newborns (24-32 wk gestational age) was assessed using early and near-term magnetic resonance imaging (MRI) studies. Red blood cell fatty acid composition was analyzed coordinated with each scan. Outcome at a mean of 33 mo corrected age was assessed using the Bayley Scales of Infant Development, 3rd edition. RESULTS: Adjusting for confounders, a 1% increase in postnatal docosahexaenoic acid (DHA) levels at early MRI was associated with 4.3-fold decreased odds of intraventricular hemorrhage, but was not associated with white matter injury or cerebellar haemorrhage. Higher DHA and lower linoleic acid (LA) levels at early MRI were associated with lower diffusivity in white matter tracts and corresponding improved developmental scores in follow-up. CONCLUSION: Higher DHA and lower LA levels in the first few weeks of life are associated with decreased intraventricular haemorrhage, improved microstructural brain development, and improved outcomes in preterm born children. Early and possibly antenatal interventions in high-risk pregnancies need to be studied for potential benefits in preterm developmental outcomes.

GenomeVISTA--an integrated software package for whole-genome alignment and visualization.

UNLABELLED: With the ubiquitous generation of complete genome assemblies for a variety of species, efficient tools for whole-genome alignment along with user-friendly visualization are critically important. Our VISTA family of tools for comparative genomics, based on algorithms for pairwise and multiple alignments of genomic sequences and whole-genome assemblies, has become one of the standard techniques for comparative analysis. Most of the VISTA programs have been implemented as Web-accessible servers and are extensively used by the biomedical community. In this manuscript, we introduce GenomeVISTA: a novel implementation that incorporates most features of the VISTA family--fast and accurate alignment, visualization capabilities, GUI and analytical tools within a stand-alone software package. GenomeVISTA thus provides flexibility and security for users who need to conduct whole-genome comparisons on their own computers. AVAILABILITY AND IMPLEMENTATION: Implemented in Perl, C/C++ and Java, the source code is freely available for download at the VISTA Web site: http://genome.lbl.gov/vista/.

Developing OCHROdb, a comprehensive quality checked database of open chromatin regions from sequencing data.

International consortia, including ENCODE, Roadmap Epigenomics, Genomics of Gene Regulation and Blueprint Epigenome have made large-scale datasets of open chromatin regions publicly available. While these datasets are extremely useful for studying mechanisms of gene regulation in disease and cell development, they only identify open chromatin regions in individual samples. A uniform comparison of accessibility of the same regulatory sites across multiple samples is necessary to correlate open chromatin accessibility and expression of target genes across matched cell types. Additionally, although replicate samples are available for majority of cell types, a comprehensive replication-based quality checking of individual regulatory sites is still lacking. We have integrated 828 DNase-I hypersensitive sequencing samples, which we have uniformly processed and then clustered their regulatory regions across all samples. We checked the quality of open-chromatin regions using our replication test. This has resulted in a comprehensive, quality-checked database of Open CHROmatin (OCHROdb) regions for 194 unique human cell types and cell lines which can serve as a reference for gene regulatory studies involving open chromatin. We have made this resource publicly available: users can download the whole database, or query it for their genomic regions of interest and visualize the results in an interactive genome browser.

White matter injury predicts disrupted functional connectivity and microstructure in very preterm born neonates.

OBJECTIVE: To determine whether the spatial extent and location of early-identified punctate white matter injury (WMI) is associated with regionally-specific disruptions in thalamocortical-connectivity in very-preterm born neonates. METHODS: 37 very-preterm born neonates (median gestational age: 28.1 weeks; interquartile range [IQR]: 27-30) underwent early MRI (median age 32.9 weeks; IQR: 32-35), and WMI was identified in 13 (35%) neonates. Structural T1-weighted, resting-state functional Magnetic Resonance Imaging (rs-fMRI, n = 34) and Diffusion Tensor Imaging (DTI, n = 31) sequences were acquired using 3 T-MRI. A probabilistic map of WMI was developed for the 13 neonates demonstrating brain injury. A neonatal atlas was applied to the WMI maps, rs-fMRI and DTI analyses to extract volumetric, functional and microstructural data from regionally-specific brain areas. Associations of thalamocortical-network strength and alterations in fractional anisotropy (FA, a measure of white-matter microstructure) with WMI volume were assessed in general linear models, adjusting for age at scan and cerebral volumes. RESULTS: WMI volume in the superior (ß = -0.007; p = .02) and posterior corona radiata (ß = -0.01; p = .01), posterior thalamic radiations (ß = -0.01; p = .005) and superior longitudinal fasciculus (ß = -0.02; p = .001) was associated with reduced connectivity strength between thalamus and parietal resting-state networks. WMI volume in the left (ß = -0.02; p = .02) and right superior corona radiata (ß = -0.03; p = .008), left posterior corona radiata (ß = -0.03; p = .01), corpus callosum (ß = -0.11; p < .0001) and right superior longitudinal fasciculus (ß = -0.02; p = .02) was associated with functional connectivity strength between thalamic and sensorimotor networks. Increased WMI volume was also associated with decreased FA values in the corpus callosum (ß = -0.004, p = .015). CONCLUSIONS: Regionally-specific alterations in early functional and structural network complexity resulting from WMI may underlie impaired outcomes.

Nutrient Intake in the First Two Weeks of Life and Brain Growth in Preterm Neonates.

BACKGROUND: Optimizing early nutritional intake in preterm neonates may promote brain health and neurodevelopment through enhanced brain maturation. Our objectives were (1) to determine the association of energy and macronutrient intake in the first 2 weeks of life with regional and total brain growth and white matter (WM) maturation, assessed by 3 serial MRI scans in preterm neonates; (2) to examine how critical illness modifies this association; and (3) to investigate the relationship with neurodevelopmental outcomes. METHODS: Forty-nine preterm neonates (21 boys, median [interquartile range] gestational age: 27.6 [2.3] weeks) were scanned serially at the following median postmenstrual weeks: 29.4, 31.7, and 41. The total brain, basal nuclei, and cerebellum were semiautomatically segmented. Fractional anisotropy was extracted from diffusion tensor imaging data. Nutritional intake from day of life 1 to 14 was monitored and clinical factors were collected. RESULTS: Greater energy and lipid intake predicted increased total brain and basal nuclei volumes over the course of neonatal care to term-equivalent age. Similarly, energy and lipid intake were significantly associated with fractional anisotropy values in selected WM tracts. The association of ventilation duration with smaller brain volumes was attenuated by higher energy intake. Brain growth predicted psychomotor outcome at 18 months' corrected age. CONCLUSIONS: In preterm neonates, greater energy and enteral feeding during the first 2 weeks of life predicted more robust brain growth and accelerated WM maturation. The long-lasting effect of early nutrition on neurodevelopment may be mediated by enhanced brain growth. Optimizing nutrition in preterm neonates may represent a potential avenue to mitigate the adverse brain health consequences of critical illness.

Severe retinopathy of prematurity predicts delayed white matter maturation and poorer neurodevelopment.

OBJECTIVE: To determine whether severe retinopathy of prematurity (ROP) is associated with (1) abnormal white matter maturation and (2) neurodevelopmental outcomes at 18 months' corrected age (CA) compared with neonates without severe ROP. DESIGN: We conducted a prospective longitudinal cohort of extremely preterm neonates born 24-28 weeks' gestational age recruited between 2006 and 2013 with brain MRIs obtained both early in life and at term-equivalent age. Severe ROP was defined as ROP treated with retinal laser photocoagulation. Using diffusion tensor imaging and tract-based spatial statistics (TBSS), white matter maturation was assessed by mean fractional anisotropy (FA) in seven predefined regions of interest. Neurodevelopmental outcomes were assessed with Bayley Scales of Infant and Toddler Development-III (Bayley-III) composite scores at 18 months' CA. Subjects were compared using Fisher's exact, Kruskal-Wallis and generalised estimating equations. SETTING: Families were recruited from the neonatal intensive care unit at BC Women's Hospital. PATIENTS: Of 98 extremely preterm neonates (median: 26.0 weeks) assessed locally for ROP, 19 (19%) had severe ROP and 83 (85%) were assessed at 18 months' CA. RESULTS: Severe ROP was associated with lower FA in the posterior white matter, and with decreased measures of brain maturation in the optic radiations, posterior limb of the internal capsule (PLIC) and external capsule on TBSS. Bayley-III cognitive and motor scores were lower in infants with severe ROP. CONCLUSIONS: Severe ROP is associated with maturational delay in the optic radiations, PLIC, external capsule and posterior white matter, housing the primary visual and motor pathways, and is associated with poorer cognitive and motor outcomes at 18 months' CA.

Prioritizing Clinically Relevant Copy Number Variation from Genetic Interactions and Gene Function Data.

It is becoming increasingly necessary to develop computerized methods for identifying the few disease-causing variants from hundreds discovered in each individual patient. This problem is especially relevant for Copy Number Variants (CNVs), which can be cheaply interrogated via low-cost hybridization arrays commonly used in clinical practice. We present a method to predict the disease relevance of CNVs that combines functional context and clinical phenotype to discover clinically harmful CNVs (and likely causative genes) in patients with a variety of phenotypes. We compare several feature and gene weighing systems for classifying both genes and CNVs. We combined the best performing methodologies and parameters on over 2,500 Agilent CGH 180k Microarray CNVs derived from 140 patients. Our method achieved an F-score of 91.59%, with 87.08% precision and 97.00% recall. Our methods are freely available at https://github.com/compbio-UofT/cnv-prioritization. Our dataset is included with the supplementary information.

Expansion and diversification of the Populus R2R3-MYB family of transcription factors.

The R2R3-MYB proteins comprise one of the largest families of transcription factors in plants. R2R3-MYB family members regulate plant-specific processes, such as the elaboration of specialized cell types, including xylem, guard cells, trichomes, and root hairs, and the biosynthesis of specialized branches of metabolism, including phenylpropanoid biosynthesis. As such, R2R3-MYB family members are hypothesized to contribute to the emergence of evolutionary innovations that have arisen in specific plant lineages. As a first step in determining the role played by R2R3-MYB family members in the emergence of lineage-specific innovations in the genus Populus, the entire Populus trichocarpa R2R3-MYB family was characterized. The Populus R2R3-MYB complement is much larger than that found in other angiosperms with fully sequenced genomes. Phylogenetic analyses, together with chromosome placement, showed that the expansion of the Populus R2R3-MYB family was not only attributable to whole genome duplication but also involved selective expansion of specific R2R3-MYB clades. Expansion of the Populus R2R3-MYB family prominently involved members with expression patterns that suggested a role in specific components of Populus life history, including wood formation and reproductive development. An expandable compendium of microarray-based expression data (PopGenExpress) and associated Web-based tools were developed to better enable within- and between-species comparisons of Populus R2R3-MYB gene expression. This resource, which includes intuitive graphic visualization of gene expression data across multiple tissues, organs, and treatments, is freely available to, and expandable by, scientists wishing to better understand the genome biology of Populus, an ecologically dominant and economically important forest tree genus.

Elucidating the germination transcriptional program using small molecules.

The transition from seed to seedling is mediated by germination, a complex process that starts with imbibition and completes with radicle emergence. To gain insight into the transcriptional program mediating germination, previous studies have compared the transcript profiles of dry, dormant, and germinating after-ripened Arabidopsis (Arabidopsis thaliana) seeds. While informative, these approaches did not distinguish the transcriptional responses due to imbibition, shifts in metabolism, or breaking of dormancy from those triggered by the initiation of germination. In this study, three mechanistically distinct small molecules that inhibit Arabidopsis seed germination (methotrexate, 2, 4-dinitrophenol, and cycloheximide) were identified using a small-molecule screen and used to probe the germination transcriptome. Germination-responsive transcripts were defined as those with significantly altered transcript abundance across all inhibitory treatments with respect to control germinating seeds, using data from ATH1 microarrays. This analysis identified numerous germination regulators as germination responsive, including the DELLA proteins GAI, RGA, and RGL3, the abscisic acid-insensitive proteins ABI4, ABI5, ABI8, and FRY1, and the gibberellin receptor GID1A. To help visualize these and other publicly available seed microarray data, we designed a seed mRNA expression browser using the electronic Fluorescent Pictograph platform. An overall decrease in gene expression and a 5-fold greater number of transcripts identified as statistically down-regulated in drug-inhibited seeds point to a role for mRNA degradation or turnover during seed germination. The genes identified in our study as responsive to germination define potential uncharacterized regulators of this process and provide a refined transcriptional signature for germinating Arabidopsis seeds.