Systematic reanalysis of genomic data improves quality of variant interpretation.

As genomic sequencing expands, so does our knowledge of the link between genetic variation and disease. Deeper catalogs of variant frequencies improve identification of benign variants, while sequencing affected individuals reveals disease-associated variation. Accumulation of human genetic data thus makes reanalysis a means to maximize the benefits of clinical sequencing. We implemented pipelines to systematically reassess sequencing data from 494 individuals with developmental disability. Reanalysis yielded pathogenic or likely pathogenic (P/LP) variants that were not initially reported in 23 individuals, 6 described here, comprising a 16% increase in P/LP yield. We also downgraded 3 LP and 6 variants of uncertain significance (VUS) due to updated population frequency data. The likelihood of identifying a new P/LP variant increased over time, as ~22% of individuals who did not receive a P/LP variant at their original analysis subsequently did after 3 years. We show here that reanalysis and data sharing increase the diagnostic yield and accuracy of clinical sequencing.

GABA estimation in the brains of children on the autism spectrum: measurement precision and regional cortical variation.

(1)H magnetic resonance spectroscopy ((1)H MRS) and spectral editing methods, such as MEGA-PRESS, allow researchers to investigate metabolite and neurotransmitter concentrations in-vivo. Here we address the utilization of (1)H MRS for the investigation of GABA concentrations in the ASD brain, in three locations; motor, visual and auditory areas. An initial repeatability study (5 subjects, 5 repeated measures separated by ~5days on average) indicated no significant effect of reference metabolite choice on GABA quantitation (p>0.6). Coefficients of variation for GABA+/NAA, GABA+/Cr and GABA+/Glx were all of the order of 9-11%. Based on these findings, we investigated creatine-normalized GABA+ ratios (GABA+/Cr) in a group of (N=17) children with autism spectrum disorder (ASD) and (N=17) typically developing children (TD) for Motor, Auditory and Visual regions of interest (ROIs). Linear regression analysis of gray matter (GM) volume changes (known to occur with development) revealed a significant decrease of GM volume with Age for Motor (F(1,30)=17.92; p<0.001) and Visual F(1,16)=14.41; p<0.005 but not the Auditory ROI (p=0.55). Inspection of GABA+/Cr changes with Age revealed a marginally significant change for the Motor ROI only (F(1,30)=4.11; p=0.054). Subsequent analyses were thus conducted for each ROI separately using Age and GM volume as covariates. No group differences in GABA+/Cr were observed for the Visual ROI between TD vs. ASD children. However, the Motor and Auditory ROI showed significantly reduced GABA+/Cr in ASD (Motor p<0.05; Auditory p<0.01). The mean deficiency in GABA+/Cr from the Motor ROI was approximately 11% and Auditory ROI was approximately 22%. Our novel findings support the model of regional differences in GABA+/Cr in the ASD brain, primarily in Auditory and to a lesser extent Motor but not Visual areas.

Utility of long-read sequencing for All of Us.

The All of Us (AoU) initiative aims to sequence the genomes of over one million Americans from diverse ethnic backgrounds to improve personalized medical care. In a recent technical pilot, we compare the performance of traditional short-read sequencing with long-read sequencing in a small cohort of samples from the HapMap project and two AoU control samples representing eight datasets. Our analysis reveals substantial differences in the ability of these technologies to accurately sequence complex medically relevant genes, particularly in terms of gene coverage and pathogenic variant identification. We also consider the advantages and challenges of using low coverage sequencing to increase sample numbers in large cohort analysis. Our results show that HiFi reads produce the most accurate results for both small and large variants. Further, we present a cloud-based pipeline to optimize SNV, indel and SV calling at scale for long-reads analysis. These results lead to widespread improvements across AoU.

Novel rare variants in congenital cardiac arrhythmia genes are frequent in drug-induced torsades de pointes.

Marked prolongation of the QT interval and polymorphic ventricular tachycardia following medication (drug-induced long QT syndrome, diLQTS) is a severe adverse drug reaction (ADR) that phenocopies congenital long QT syndrome (cLQTS) and is one of the leading causes for drug withdrawal and relabeling. We evaluated the frequency of rare non-synonymous variants in genes contributing to the maintenance of heart rhythm in cases of diLQTS using targeted capture coupled to next-generation sequencing. Eleven of 31 diLQTS subjects (36%) carried a novel missense mutation in genes with known congenital arrhythmia associations or with a known cLQTS mutation. In the 26 Caucasian subjects, 23% carried a highly conserved rare variant predicted to be deleterious to protein function in these genes compared with only 2-4% in public databases (P<0.003). We conclude that the rare variation in genes responsible for congenital arrhythmia syndromes is frequent in diLQTS. Our findings demonstrate that diLQTS is a pharmacogenomic syndrome predisposed by rare genetic variants.

Expression and sequence analysis of the mouse adenine nucleotide translocase 1 and 2 genes.

Only two isoforms of the adenine nucleotide translocase (Ant) protein have been identified in mouse, as opposed to the three in humans. To determine whether the homologous mouse and human proteins share similar patterns of expression, Northern and Western analyses were performed on several mouse tissues. Mouse Ant1 is expressed at high levels in skeletal muscle and heart, similar to human ANT1. Mouse Ant2 is strongly expressed in all tissues but muscle, in marked contrast to human ANT2. To investigate the molecular basis of these differences, we cloned and sequenced the genomic loci of mouse Ant1 and Ant2, and compared them to the three human ANT loci. The mouse and human ANT1 and ANT2 genes showed substantial homology starting about 300 base pairs (bp) 5' to the coding region and continuing through the 3' untranslated region (UTR). Repeats constituted 32% of 15kb of Ant1 sequence and 36% of the 27kb of Ant2 sequence and included SINEs, LINEs and LTR elements. The core promoters of the mouse and human ANT1 and ANT2 genes are very similar. However, the mouse Ant1 gene lacks the upstream OXBOX and REBOX elements found in human ANT1 genes, thought to be important for muscle-specific expression. The mouse Ant2 gene, like human ANT2, has an upstream GRBOX, yet this element is not associated with suppression of transcription, as hypothesized for human ANT2. These discrepancies indicate that additional studies will be required to fully understand the transcriptional regulation of both Ant1 and Ant2.

Practice parameter: screening and diagnosis of autism: report of the Quality Standards Subcommittee of the American Academy of Neurology and the Child Neurology Society.

Autism is a common disorder of childhood, affecting 1 in 500 children. Yet, it often remains unrecognized and undiagnosed until or after late preschool age because appropriate tools for routine developmental screening and screening specifically for autism have not been available. Early identification of children with autism and intensive, early intervention during the toddler and preschool years improves outcome for most young children with autism. This practice parameter reviews the available empirical evidence and gives specific recommendations for the identification of children with autism. This approach requires a dual process: 1) routine developmental surveillance and screening specifically for autism to be performed on all children to first identify those at risk for any type of atypical development, and to identify those specifically at risk for autism; and 2) to diagnose and evaluate autism, to differentiate autism from other developmental disorders.

Lead poisoning in a child with a gunshot wound.

Lead poisoning has infrequently resulted from gunshot wounds with retained lead particles in adults. This has not been previously found in children. The case of an 8-year-old boy in whom lead poisoning developed soon after a gunshot wound is reported. The child had no symptoms directly related to lead poisoning, but he received chelation therapy. The case demonstrates the need to consider lead poisoning in children with retained particles following gunshot wounds.

Proficiency testing materials for pH and blood gases. The California Thoracic Society experience.

The California Thoracic Society Blood Gas Proficiency Testing Program distributed ampules from three separate lots of quality control products every three months as unknowns to participating clinical (survey) laboratories and ten selected reference laboratories. For eight quarters, aqueous buffers were distributed. For each lot, PCO2 and pH measurements varied within narrow ranges between laboratories. Concurrently, the PO2 measurements varied widely between reference laboratories as well as survey laboratories, but varied minimally when repeatedly assessed on each reference laboratory machine. Change to a fluorocarbon-containing emulsion as a testing medium resulted in a significant reduction in within model and overall variability for PO2. We attribute this reduction in variability to the higher O2 content and decreased temperature sensitivity for PO2 of the fluorocarbon-containing emulsion. Because we have no evidence that the magnitude of the interinstrument differences in PO2 found with these materials would be found with fresh human blood we recommend that regulatory agencies use the results of proficiency testing for PO2 cautiously.

The screening and diagnosis of autistic spectrum disorders.

The Child Neurology Society and American Academy of Neurology recently proposed to formulate Practice Parameters for the Diagnosis and Evaluation of Autism for their memberships. This endeavor was expanded to include representatives from nine professional organizations and four parent organizations, with liaisons from the National Institutes of Health. This document was written by this multidisciplinary Consensus Panel after systematic analysis of over 2,500 relevant scientific articles in the literature. The Panel concluded that appropriate diagnosis of autism requires a dual-level approach: (a) routine developmental surveillance, and (b) diagnosis and evaluation of autism. Specific detailed recommendations for each level have been established in this document, which are intended to improve the rate of early suspicion and diagnosis of, and therefore early intervention for, autism.

Pediatric assessment of the child with developmental delay.

With the recent mandate for early intervention services, the pediatrician is more involved than ever in the identification and evaluation of children with developmental disabilities. Developmental surveillance at routine visits and listening to parental concerns are crucial in the early diagnosis of developmental delay so that therapeutic interventions can be provided at a time when there is a good chance of decreasing disability and family stress. Medical evaluations and consultations must be prudent and based on a thorough history and physical. The pediatrician must have the knowledge and skills to coordinate medical care and to counsel and provide support to the child and family as they receive habilitative services and come to terms with the developmental diagnoses.

Environmental factors influencing weaning of a young child from mechanical ventilator support.

We examined the contribution of physiologic and environmental variables to the process of weaning a child with chronic respiratory failure from mechanical ventilation support. Surveillance measures, e.g., blood oxygenation, were obtained from a 6-year-old child with multiple medical and developmental disorders who received three different rates (24, 22, and 20 tidal volumes per minute) of intermittent mechanical ventilation. Direct observations were used to calculate rates of aberrant behavior, e.g., aggression toward self, for task versus play settings within the intermittent mechanical ventilation rates. Rates of aberrant behavior and adult responses were tabulated from videotaped observations for task, attention, and no attention settings. The greatest rate of aberrant behavior occurred during tasks compared with play activities, regardless of whether attention was provided while playing. Adults also responded more often to aberrant behaviors during task versus play conditions. Clinical implications are discussed concerning the inclusion of developmental and behavioral variables during weaning from mechanical ventilation.

Impaired emotional health in children with mild reading disability.

Children with reading disabilities are at risk for emotional difficulties. There is some evidence that reading-disabled children with high socioeconomic status may be at risk of having low self-esteem. We conducted a preliminary study of the impact of reading disability on children's self-esteem and emotional health in a select group of mildly impaired reading disabled children with well-educated parents who were well informed about reading disabilities. We interviewed 28 healthy, preadolescent boys and girls with mean reading delay of 9.0 months and 39 comparable nonreading disabled children who formed the control group. Children and parents completed the Harter's Self-Perception Profile. Parents completed the Achenbach Child Behavior Checklist and Rand Mental Health Survey. We found that these mildly impaired reading-disabled children were more anxious and less happy than were nondisabled students despite having well-informed, well-educated parents (mean level of education = 17.0 years). Reading-disabled children considered themselves to be less competent scholastically, and their parents rated them as less competent than nondisabled children on all measures of self-esteem. These well-educated mothers tended to underrate their child's perceived scholastic competence. Controlling for behavior problems, Achenbach Child Behavior Checklist scores for social competence were lower for reading-disabled children. These findings suggest that the mildly impaired reading-disabled children with high socioeconomic status are at risk for low self-esteem and poor emotional health.

Left hemisphere diffusivity of the arcuate fasciculus: influences of autism spectrum disorder and language impairment.

BACKGROUND AND PURPOSE: There has been much discussion whether brain abnormalities associated with specific language impairment and autism with language impairment are shared or are disorder specific. Although white matter tract abnormalities are observed in both specific language impairment and autism spectrum disorders, the similarities and differences in the white matter abnormalities in these 2 disorders have not been fully determined. MATERIALS AND METHODS: Diffusion tensor imaging diffusion parameters of the arcuate fasciculus were measured in 14 children with specific language impairment as well as in 16 children with autism spectrum disorder with language impairment, 18 with autism spectrum disorder without language impairment, and 25 age-matched typically developing control participants. RESULTS: Language impairment and autism spectrum disorder both had (elevating) main effects on mean diffusivity of the left arcuate fasciculus, initially suggesting a shared white matter substrate abnormality. Analysis of axial and radial diffusivity components, however, indicated that autism spectrum disorder and language impairment differentially affect white matter microstructural properties, with a main effect of autism spectrum disorder on axial diffusivity and a main effect of language impairment on radial diffusivity. CONCLUSIONS: Although white matter abnormalities appear similar in language impairment and autism spectrum disorder when examining broad white matter measures, a more detailed analysis indicates different mechanisms for the white matter microstructural anomalies associated with language impairment and autism spectrum disorder.

Elevated mean diffusivity in the left hemisphere superior longitudinal fasciculus in autism spectrum disorders increases with more profound language impairment.

BACKGROUND AND PURPOSE: Language impairments are observed in a subset of individuals with ASD. To examine microstructural brain white matter features associated with language ability in ASD, we measured the DTI parameters of language-related white matter tracts (SLF) as well as non-language-related white matter tracts (CST) in children with ASD/+LI and ASD/-LI) and in TD. MATERIALS AND METHODS: Eighteen children with ASD/-LI (age range, 6.7-17.5 years), 17 with ASD/+LI (age range, 6.8-14.8 years), and 25 TD (age range, 6.5-18 years) were evaluated with DTI and tractography. Primary DTI parameters considered for analysis were MD and FA. RESULTS: There was a main effect of diagnostic group on age-corrected MD (P < .05) with ASD/+LI significantly elevated compared with TD. This was most pronounced for left hemisphere SLF fiber tracts and for the temporal portion of the SLF. There was significant negative correlation between left hemisphere SLF MD values and the clinical assessment of language ability. There was no main effect of diagnostic group or diagnostic group X hemisphere interaction for FA. Although there was a main effect of diagnostic group on values of MD in the CST, this did not survive hemispheric subanalysis. CONCLUSIONS: Abnormal DTI parameters (specifically significantly elevated MD values in ASD) of the SLF appear to be associated with language impairment in ASD. These elevations are particularly pronounced in the left cerebral hemisphere, in the temporal portion of the SLF, and in children with clinical language impairment.

A microarray analysis of temporal gene expression profiles in thermally injured human skin.

Partial-thickness burns incite a multitude of responses which eventually culminate in cutaneous wound repair. We hypothesized that these events would evoke extensive alterations in gene expression thereby orchestrating the complexity of spatial and temporal events that characterize "normal" human wound healing. In the present study, gene expression from partial-thickness areas at defined temporal periods (1-3 days, 4-6 days, and 7-18 days) after injury were compared to normal non-wounded skin. Gene alterations proved extensive (2286 genes). Statistically significant alterations were noted among increased and decreased genes expressed in the three different temporal groupings. Our foundational data (based on samples from 45 individuals) provide a comprehensive molecular gene expression portrait of the cutaneous reparative responses that are initiated during the first 17 days after injury. Our efforts also represent an initial endeavor to move beyond the historically defined "morphological phases" of wound repair toward reporting molecular clues that define the temporal sequence of healing in human subjects. Further analysis of genes that are either affected or remain not affected following injury to normal skin is expected to identify potential targets for therapeutic augmentation or silencing.

Mechanism of arterial hypoxemia following pulmonary thromboembolism in dogs.

Mongrel dogs (29) were anesthetized, paralyzed, and ventilated at a constant minute volume. AaD02 breathing air and 100% O2, venous admixture breathing air (Qva/Qt) and 100% O2 (Qs/Qt), single-breath diffusing capacity for CO (DLCO), and total pulmonary resistance (RL) and pulmonary compliance (CL) were measured before and after pulmonary embolization with autologus in vivo venous thrombi. Nine dogs were heparinized before embolization. In the 20 nonheparinized dogs AaDo2 breathing air increased from 11 to 26 mmHg, Qva/Qt from 4 to 22%, and Qs/At from 5 to 8%. DLCO decreased 24%, RL increased 43%, and CL fell 30%. In the nine heparinized dogs AaDo2 breathing air increased from 8 to 13 mmHg and Qva/Qt from 3 to 8%; Qs/Qt did not change. DLCO decreased 31%; RL and CL did not change significantly. The increase in Qva/Qt of 5% in the heparinized dogs was significantly less (P smaller than 0.001) than the increase of 18% in the nonheparinized dogs. These findings suggest that arterial hypoxemia following thromboembolism is due to ventilation-perfusion inequality caused by changes in lung mechanics.