Diagnosis of genetic diseases in seriously ill children by rapid whole-genome sequencing and automated phenotyping and interpretation.

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.

Plasma genetic and genomic abnormalities predict treatment response and clinical outcome in advanced prostate cancer.

Liquid biopsies, examinations of tumor components in body fluids, have shown promise for predicting clinical outcomes. To evaluate tumor-associated genomic and genetic variations in plasma cell-free DNA (cfDNA) and their associations with treatment response and overall survival, we applied whole genome and targeted sequencing to examine the plasma cfDNAs derived from 20 patients with advanced prostate cancer. Sequencing-based genomic abnormality analysis revealed locus-specific gains or losses that were common in prostate cancer, such as 8q gains, AR amplifications, PTEN losses and TMPRSS2-ERG fusions. To estimate tumor burden in cfDNA, we developed a Plasma Genomic Abnormality (PGA) score by summing the most significant copy number variations. Cox regression analysis showed that PGA scores were significantly associated with overall survival (p < 0.04). After androgen deprivation therapy or chemotherapy, targeted sequencing showed significant mutational profile changes in genes involved in androgen biosynthesis, AR activation, DNA repair, and chemotherapy resistance. These changes may reflect the dynamic evolution of heterozygous tumor populations in response to these treatments. These results strongly support the feasibility of using non-invasive liquid biopsies as potential tools to study biological mechanisms underlying therapy-specific resistance and to predict disease progression in advanced prostate cancer.

Genetic variations in EGFR and ERBB4 increase susceptibility to cervical cancer.

OBJECTIVES: Inherited genetic variability contributes to susceptibility to cervical cancer. We investigated the association of single nucleotide polymorphisms (SNPs) in the human epidermal growth factor receptor (ERBB) family with cervical cancer. METHODS: We used the transmission disequilibrium test (TDT) to look for excessive transmission of tag single nucleotide polymorphisms (tSNPs) in ERBB family members EGFR, ERBB2, ERBB3, and ERBB4 in a large sample of women with invasive and in situ cervical cancer and their biological parents (628 trios). The study used a discovery set of trios (244) analyzed by Illumina GoldenGate in which SNPs reaching a P<.05 were re-tested by TaqMan in the combined set of 628. We also explored collaborative effects of different ERBB alleles. RESULTS: Based on single SNP TDT tests we identified 16 significant SNPs in the discover stage and six of 14 SNPs that could be assayed by TaqMan were significantly overtransmitted in women with cervical cancer in the combined replication set. Four SNPs were located in intron 1 of EGFR and two SNPs in intron 24 of ERBB4. The EGFR variants are located near multiple enhancers, silencers, and the previously identified functional common polymorphisms in intron 1. CONCLUSIONS: Our data provide evidence for the involvement of intron 1 EGFR variants and intron 24 ERBB4 variants in modulating risk for the development of in situ and invasive cervical cancer. These variants should be examined in additional populations and functional studies would be needed to confirm this hypothesis.

Genome-wide identification of HrpL-regulated genes in the necrotrophic phytopathogen Dickeya dadantii 3937.

BACKGROUND: Dickeya dadantii is a necrotrophic pathogen causing disease in many plants. Previous studies have demonstrated that the type III secretion system (T3SS) of D. dadantii is required for full virulence. HrpL is an alternative sigma factor that binds to the hrp box promoter sequence of T3SS genes to up-regulate their expression. METHODOLOGY/PRINCIPAL FINDINGS: To explore the inventory of HrpL-regulated genes of D. dadantii 3937 (3937), transcriptome profiles of wild-type 3937 and a hrpL mutant grown in a T3SS-inducing medium were examined. Using a cut-off value of 1.5, significant differential expression was observed in sixty-three genes, which are involved in various cellular functions such as type III secretion, chemotaxis, metabolism, regulation, and stress response. A hidden Markov model (HMM) was used to predict candidate hrp box binding sites in the intergenic regions of 3937, including the promoter regions of HrpL-regulated genes identified in the microarray assay. In contrast to biotrophic phytopathgens such as Pseudomonas syringae, among the HrpL up-regulated genes in 3937 only those within the T3SS were found to contain a hrp box sequence. Moreover, direct binding of purified HrpL protein to the hrp box was demonstrated for hrp box-containing DNA fragments of hrpA and hrpN using the electrophoretic mobility shift assay (EMSA). In this study, a putative T3SS effector DspA/E was also identified as a HrpL-upregulated gene, and shown to be translocated into plant cells in a T3SS-dependent manner. CONCLUSION/SIGNIFICANCES: We provide the genome-wide study of HrpL-regulated genes in a necrotrophic phytopathogen (D. dadantii 3937) through a combination of transcriptomics and bioinformatics, which led to identification of several effectors. Our study indicates the extent of differences for T3SS effector protein inventory requirements between necrotrophic and biotrophic pathogens, and may allow the development of different strategies for disease control for these different groups of pathogens.

Transcriptional profiling of methyltransferase genes during growth of Methanosarcina mazei on trimethylamine.

The genomic expression patterns of Methanosarcina mazei growing with trimethylamine were measured in comparison to those of cells grown with methanol. We identified a total of 72 genes with either an increased level (49 genes) or a decreased level (23 genes) of mRNA during growth on trimethylamine with methanol-grown cells as the control. Major differences in transcript levels were observed for the mta, mtb, mtt, and mtm genes, which encode enzymes involved in methane formation from methanol and trimethylamine, respectively. Other differences in mRNA abundance were found for genes encoding enzymes involved in isopentenyl pyrophosphate synthesis and in the formation of aromatic amino acids, as well as a number of proteins with unknown functions. The results were verified by in-depth analysis of methyltransferase genes using specific primers for real-time quantitative reverse transcription-PCR (RT-PCR). The monitored transcript levels of genes encoding corrinoid proteins involved in methyl group transfer from methylated C(1) compounds (mtaC, mtbC, mttC, and mtmC) indicated increased amounts of mRNA from the mtaBC1, mtaBC2, and mtaBC3 operons in methanol-grown cells, whereas mRNA of the mtb1-mtt1 operon was found in high concentrations during trimethylamine consumption. The genes of the mtb1-mtt1 operon encode methyltransferases that are responsible for sequential demethylation of trimethylamine. The analysis of product formation of trimethylamine-grown cells at different optical densities revealed that large amounts of dimethylamine and monomethylamine were excreted into the medium. The intermediate compounds were consumed only in the very late exponential growth phase. RT-PCR analysis of key genes involved in methanogenesis led to the conclusion that M. mazei is able to adapt to changing trimethylamine concentrations and the consumption of intermediate compounds. Hence, we assume that the organism possesses a regulatory network for optimal substrate utilization.

Global transcriptional analysis of Methanosarcina mazei strain Gö1 under different nitrogen availabilities.

Certain archaeal species can fix molecular nitrogen under nitrogen limiting conditions although little is known about this process at either the genetic or molecular level. To address this on a genome-wide scale, transcriptional analysis was performed on the model methanogen Methanosarcina mazei strain Gö1 using DNA-microarrays. The genomic expression patterns for cells grown under nitrogen fixing conditions versus nitrogen sufficiency (10 mM ammonium) revealed that approximately 5% of all genes are differentially expressed. Besides a small set of genes previously known to be up-regulated under nitrogen limitation, 14 additional genes involved in nitrogen metabolism were identified plus 10 genes encoding potential transcriptional regulators, 13 genes involved in carbon metabolism, 3 genes in general stress response, 8 putative transporter genes, and an additional 21 genes with unknown function. Quantitative reverse transcriptase PCR experiments confirmed the differential expression of a subset of these genes. Promoter analysis revealed a palindromic DNA motif centered nearby the transcriptional start point for several genes up-regulated under nitrogen limitation. A bioinformatics study demonstrated the presence of this motif in the up-stream region of 52 genes genome-wide, the majority of which showed nitrogen dependent differential transcription. We therefore hypothesize that this DNA element is involved in nitrogen control in M. mazei where it may act as a binding site for a regulatory protein.

MprAB is a stress-responsive two-component system that directly regulates expression of sigma factors SigB and SigE in Mycobacterium tuberculosis.

The genetic mechanisms mediating the adaptation of Mycobacterium tuberculosis within the host are poorly understood. The best-characterized regulatory systems in this organism include sigma factors and two-component signal transduction systems. mprAB is a two-component system required by M. tuberculosis for growth in vivo during the persistent stage of infection. In this report, we demonstrate that MprAB is stress responsive and regulates the expression of numerous stress-responsive genes in M. tuberculosis. With DNA microarrays and quantitative real-time reverse transcription-PCR, genes regulated by MprA in M. tuberculosis that included two stress-responsive sigma factors were identified. Response regulator MprA bound to conserved motifs in the upstream regions of both sigB and sigE in vitro and regulated the in vivo expression of sigB and sigE in M. tuberculosis. In addition, mprA itself was induced following exposure to stress, establishing a direct role for this regulatory system in stress response pathways of M. tuberculosis. Induction of mprA and sigE by MprA in response to stress was mediated through the cognate sensor kinase MprB and required expression of the extracytoplasmic loop domain. These results provide the first evidence that recognition of and adaptation to specific stress in M. tuberculosis are mediated through activation of a two-component signal transduction system that directly regulates the expression of stress-responsive determinants.

DNA microarray analysis of Methanosarcina mazei Gö1 reveals adaptation to different methanogenic substrates.

Methansarcina mazei Gö1 DNA arrays were constructed and used to evaluate the genomic expression patterns of cells grown on either of two alternative methanogenic substrates, acetate or methanol, as sole carbon and energy source. Analysis of differential transcription across the genome revealed two functionally grouped sets of genes that parallel the central biochemical pathways in, and reflect many known features of, acetate and methanol metabolism. These include the acetate-induced genes encoding acetate activating enzymes, acetyl-CoA synthase/CO dehydrogenase, and carbonic anhydrase. Interestingly, additional genes expressed at significantly higher levels during growth on acetate included two energy-conserving complexes (the Ech hydrogenase, and the A1A0-type ATP synthase). Many previously unknown features included the induction by acetate of genes coding for ferredoxins and flavoproteins, an aldehyde:ferredoxin oxidoreductase, enzymes for the synthesis of aromatic amino acids, and components of iron, cobalt and oligopeptide uptake systems. In contrast, methanol-grown cells exhibited elevated expression of genes assigned to the methylotrophic pathway of methanogenesis. Expression of genes for components of the translation apparatus was also elevated in cells grown in the methanol medium relative to acetate, and was correlated with the faster growth rate observed on the former substrate. These experiments provide the first comprehensive insight into substrate-dependent gene expression in a methanogenic archaeon. This genome-wide approach, coupled with the complementary molecular and biochemical tools, should greatly accelerate the exploration of Methanosarcina cell physiology, given the present modest level of our knowledge of these large archaeal genomes.