By informing timely targeted treatments, rapid whole-genome sequencing can improve the outcomes of seriously ill children with genetic diseases, particularly infants in neonatal and pediatric intensive care units (ICUs). The need for highly qualified professionals to decipher results, however, precludes widespread implementation. We describe a platform for population-scale, provisional diagnosis of genetic diseases with automated phenotyping and interpretation. Genome sequencing was expedited by bead-based genome library preparation directly from blood samples and sequencing of paired 100-nt reads in 15.5 hours. Clinical natural language processing (CNLP) automatically extracted children’s deep phenomes from electronic health records with 80% precision and 93% recall. In 101 children with 105 genetic diseases, a mean of 4.3 CNLP-extracted phenotypic features matched the expected phenotypic features of those diseases, compared with a match of 0.9 phenotypic features used in manual interpretation. We automated provisional diagnosis by combining the ranking of the similarity of a patient’s CNLP phenome with respect to the expected phenotypic features of all genetic diseases, together with the ranking of the pathogenicity of all of the patient’s genomic variants. Automated, retrospective diagnoses concurred well with expert manual interpretation (97% recall and 99% precision in 95 children with 97 genetic diseases). Prospectively, our platform correctly diagnosed three of seven seriously ill ICU infants (100% precision and recall) with a mean time saving of 22:19 hours. In each case, the diagnosis affected treatment. Genome sequencing with automated phenotyping and interpretation in a median of 20:10 hours may increase adoption in ICUs and, thereby, timely implementation of precise treatments.
Although transforming growth factor-beta (TGF-) signaling negatively regulates branching morphogenesis in early lung development, few studies to date have addressed the role of this family of growth factors during late lung development. We describe here that the expression, tissue localization, and activity of components of the TGF- signaling machinery are dynamically regulated during late lung development in the mouse and human. Pronounced changes in the expression and localization of the TGF- receptors Acvrl1, Tgfbr1, Tgfbr2, Tgfbr3, and endoglin, and the intracellular messengers Smad2, Smad3, Smad4, Smad6, and Smad7 were noted as mouse and human lungs progressed through the canalicular, saccular, and alveolar stages of development. TGF- signaling, assessed by phosphorylation of Smad2, was detected in the vascular and airway smooth muscle, as well as the alveolar and airway epithelium throughout late lung development. These data suggest that active TGF- signaling is required for normal late lung development. Developmental Dynamics 237:259 -269, 2008.
Breathing-disordered states, such as in obstructive sleep apnea, which are cyclical in nature, have been postulated to induce neurocognitive morbidity in both pediatric and adult populations. The oscillatory nature of intermittent hypoxia, especially when chronic, may mimic the paradigm of ischemia-reperfusion in that tissues and cells are exposed to episodes of low and high O(2) and this may lead to oxidant stress. Therefore, we decided to explore the potential contribution of oxidant stress in our intermittent hypoxia/hypercapnia animal model and the role that mitochondria might play in this stress. Neonatal mice were exposed to intermittent hypoxia/hypercapnia for 10 days and 2 wk. Combined intermittent hypoxia/hypercapnia led to a marked increase in apoptotic cell death in the cerebral cortex. Oxygen consumption studies in isolated mitochondria from intermittent hypoxia/hypercapnia-exposed brains demonstrated significant reductions in both state 4 and state 3 respiratory activities by approximately 60% and 75%, respectively. Electron paramagnetic resonance spectroscopy registered a significant increase in superoxide production during nonphosphorylating state 4 by 37%, although superoxide leakage during state 3 did not increase upon treatment. Neuronal superoxide-specific dihydroethidium oxidation was also greater in exposed animals. These studies indicate that intermittent hypoxia/hypercapnia leads to oxidative stress due to mitochondrial response within the mouse central nervous system.
Bacteria of the genusT he genus Shigella comprises 4 species, including Shigella flexneri, which is comprised of 14 different serotypes and subserotypes (2). S. flexneri strain M90T Sm is commonly used in the laboratory to understand the molecular basis of shigellosis (1,5,9).Two amplification-free libraries were constructed from 5 g of M90T Sm genomic DNA each, and the genome was sequenced using a 2-by-110-base paired-end strategy on the Illumina HiSeq2000 platform. Libraries were constructed using the NEBNext DNA sample prep master mix set 1 (NEB; part number E6040L) and barcoded in an overnight ligation. Libraries were size selected from 450 bp to 500 bp, quantified by quantitative PCR (qPCR) (Kapa BioSystems), pooled, requantified, and clustered using cBot on a single lane with TruSeq PE cluster kit v2 chemistry. A total of 129,937,458 single-end reads were filtered by demultiplexing using two indexes (AGTCGATC/CAGTCTGA) and the signal purity filter. The resulting 73,325,436 reads were trimmed to 54 bp, the point at which the mean quality score dropped below 30 (Illumina 1.5ϩ phred quality score), using the Fastx toolkit (version 0.0.13) (http://hannonlab.cshl.edu/fastx_toolkit/index .html). Half of the preprocessed reads (34,231,825 reads) were mapped onto the chromosomal sequence of S. flexneri serotype 5b strain 8401 (GenBank accession no. NC_008258) (6) using the Burrows-Wheeler aligner (BWA) (version 0.5.9) (3). A total of 28,876,862 reads (84.3%) were successfully mapped to a depth of 340ϫ. Single nucleotide polymorphism (SNP) calls and consensus sequence generation were performed using the pileup function in SAMtools (version 0.1.16) (4). The integrated genomics viewer (IGV) (version 1.5) (7) was used to examine regions of low coverage (0ϫ to 160ϫ) and/or low quality. A total of 39 positions within the single-copy region of the genome were verified by Sanger sequencing. The remaining 119 gaps were within multicopy regions, such as insertion sequences (IS), ribosomal DNAs, and the tufA gene. The GϩC content was calculated using Artemis (version 13.0) (8).The sequence of the S. flexneri M90T Sm genome covers 99.9% of the 4,574,284-bp chromosome of S. flexneri 8401 (6). A total of 1,433 SNPs and 176 indels were identified. About one-third of the SNPs (543 sites) cause nonsynonymous substitutions in S. flexneri M90T Sm. Twenty-one indels in M90T Sm cause frameshifts, and 9 indels correct 9 frame-shifted genes in S. flexneri 8401. A tandem repeat of tRNAs (tRNA-Lys and tRNA-Val) and one hypothetical protein that both do not exist in S. flexneri 8401 were found in M90T Sm. The M90T Sm genome contains 3,676 open reading frames (ORFs) (excluding IS ORFs), 99 tRNA genes, and 22 rRNA genes. The sequence has an average GϩC content of 50.9% in its entirety with a GϩC content of 52% within coding genes.The S. flexneri M90T Sm genome will enhance ongoing wholegenome comparisons across all S. flexneri serotypes for a fuller understanding of the evolution of S. flexneri and provides accurate sequence information need...
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