Raymond J. Langley

Carrier Testing for Severe Childhood Recessive Diseases by Next-Generation Sequencing

Carrier testing for 448 severe childhood recessive diseases by next-generation sequencing has good predictive value and suggests that every individual carries about three disease mutations. Shining a Light on Comprehensive Carrier Screening Although diseases inherited in a Mendelian fashion are rare, together they account for about 20% of deaths in infancy. For Mendelian diseases that are recessive (of which there are more than 1000), screening before pregnancy (preconception screening) together with genetic counseling of those carrying a mutant allele could reduce the incidence of these diseases and the suffering that they incur. In the case of Tay-Sachs disease, an incurable neurodegenerative disease of infancy, preconception screening for disease gene mutations and genetic counseling among individuals of Ashkenazi descent has reduced the incidence of this tragic disease by 90%. However, simultaneous testing for many recessive childhood diseases is costly, so, to date, screening has included just a few diseases such as Tay-Sachs disease, cystic fibrosis, and familial dysautonomia. In a new study, Kingsmore and his colleagues have combined target gene capture and enrichment, next-generation sequencing, and sophisticated bioinformatic analysis to develop a platform capable of screening several hundred DNA samples simultaneously for 448 severe recessive diseases of childhood. They demonstrate that their method is sensitive, specific, and scalable in a research setting and that it should be straightforward to automate the process. The authors report that individuals in the general population carry an average of three recessive childhood disease mutations. They also discovered that about 10% of disease mutations in commonly used databases are incorrect, suggesting that disease mutation annotations in such databases should be carefully scrutinized. The authors predict that their screening test could be made faster and more cost-effective with the advent of microdroplet polymerase chain reaction and third-generation sequencing technologies. Their study provides a proof of concept that it should be possible to introduce preconception carrier screening for many recessive pediatric disease mutations as long as the disease genes are known. Many social, legal, and societal issues need to be addressed before preconception carrier screening can be made available for the general population, and cost is still a big consideration. However, this methodology could also be applied for comprehensive screening of newborns and would allow early diagnosis and intervention for a variety of Mendelian diseases. Although it may be some time before preconception carrier testing enters the community setting, physicians, patients, parents, and genetic counselors need to discuss the impact and implications of this new technology. Of 7028 disorders with suspected Mendelian inheritance, 1139 are recessive and have an established molecular basis. Although individually uncommon, Mendelian diseases collectively account for ~20% of infant mortality and ~10% of pediatric hospitalizations. Preconception screening, together with genetic counseling of carriers, has resulted in remarkable declines in the incidence of several severe recessive diseases including Tay-Sachs disease and cystic fibrosis. However, extension of preconception screening to most severe disease genes has hitherto been impractical. Here, we report a preconception carrier screen for 448 severe recessive childhood diseases. Rather than costly, complete sequencing of the human genome, 7717 regions from 437 target genes were enriched by hybrid capture or microdroplet polymerase chain reaction, sequenced by next-generation sequencing (NGS) to a depth of up to 2.7 gigabases, and assessed with stringent bioinformatic filters. At a resultant 160× average target coverage, 93% of nucleotides had at least 20× coverage, and mutation detection/genotyping had ~95% sensitivity and ~100% specificity for substitution, insertion/deletion, splicing, and gross deletion mutations and single-nucleotide polymorphisms. In 104 unrelated DNA samples, the average genomic carrier burden for severe pediatric recessive mutations was 2.8 and ranged from 0 to 7. The distribution of mutations among sequenced samples appeared random. Twenty-seven percent of mutations cited in the literature were found to be common polymorphisms or misannotated, underscoring the need for better mutation databases as part of a comprehensive carrier testing strategy. Given the magnitude of carrier burden and the lower cost of testing compared to treating these conditions, carrier screening by NGS made available to the general population may be an economical way to reduce the incidence of and ameliorate suffering associated with severe recessive childhood disorders.

Sepsis: An integrated clinico-metabolomic model improves prediction of death in sepsis

A molecular signature, derived from integrated analysis of clinical data, the metabolome, and the proteome in prospective human studies, improved the prediction of death in patients with sepsis, potentially identifying a subset of patients who merit intensive treatment. Understanding Survival of the Fittest in Sepsis Differentiating mild infections from life-threatening ones is a complex decision that is made millions of times a year in U.S. emergency rooms. Should a patient be sent home with antibiotics and chicken soup? Or should he or she be hospitalized for intensive treatment? Sepsis—a serious infection that is associated with a generalized inflammatory response—is one of the leading causes of death. In two prospective clinical studies reported by Langley et al., patients arriving at four urban emergency departments with symptoms of sepsis were evaluated clinically and by analysis of their plasma proteome and metabolome. Survivors and nonsurvivors at 28 days were compared, and a molecular signature was detected that appeared to differentiate these outcomes—even as early as the time of hospital arrival. The signature was part of a large set of differences between these groups, showing that better energy-producing fatty acid catabolism was associated with survival of the fittest in sepsis. A test developed from the signature was able to predict sepsis survival and nonsurvival reproducibly and better than current methods. This test could help to make all important decisions in the emergency room more accurate. Sepsis is a common cause of death, but outcomes in individual patients are difficult to predict. Elucidating the molecular processes that differ between sepsis patients who survive and those who die may permit more appropriate treatments to be deployed. We examined the clinical features and the plasma metabolome and proteome of patients with and without community-acquired sepsis, upon their arrival at hospital emergency departments and 24 hours later. The metabolomes and proteomes of patients at hospital admittance who would ultimately die differed markedly from those of patients who would survive. The different profiles of proteins and metabolites clustered into the following groups: fatty acid transport and β-oxidation, gluconeogenesis, and the citric acid cycle. They differed consistently among several sets of patients, and diverged more as death approached. In contrast, the metabolomes and proteomes of surviving patients with mild sepsis did not differ from survivors with severe sepsis or septic shock. An algorithm derived from clinical features together with measurements of five metabolites predicted patient survival. This algorithm may help to guide the treatment of individual patients with sepsis.

T Cells Express α7-Nicotinic Acetylcholine Receptor Subunits That Require a Functional TCR and Leukocyte-Specific Protein Tyrosine Kinase for Nicotine-Induced Ca2+ Response

Acute and chronic effects of nicotine on the immune system are usually opposite; acute treatment stimulates while chronic nicotine suppresses immune and inflammatory responses. Nicotine acutely raises intracellular calcium ([Ca2+]i) in T cells, but the mechanism of this response is unclear. Nicotinic acetylcholine receptors (nAChRs) are present on neuronal and non-neuronal cells, but while in neurons, nAChRs are cation channels that participate in neurotransmission; their structure and function in nonexcitable cells are not well-defined. In this communication, we present evidence that T cells express α7-nAChRs that are critical in increasing [Ca2+]i in response to nicotine. Cloning and sequencing of the receptor from human T cells showed a full-length transcript essentially identical to the neuronal α7-nAChR subunit (>99.6% homology). These receptors are up-regulated and tyrosine phosphorylated by treatment with nicotine, anti-TCR Abs, or Con A. Furthermore, knockdown of the α7-nAChR subunit mRNA by RNA interference reduced the nicotine-induced Ca2+ response, but unlike the neuronal receptor, α-bungarotoxin and methyllycaconitine not only failed to block, but also actually raised [Ca2+]i in T cells. The nicotine-induced release of Ca2+ from intracellular stores in T cells did not require extracellular Ca2+, but, similar to the TCR-mediated Ca2+ response, required activation of protein tyrosine kinases, a functional TCR/CD3 complex, and leukocyte-specific tyrosine kinase. Moreover, CD3ζ and α7-nAChR coimmunoprecipitated with anti-CD3ζ or anti-α7-nAChR Abs. These results suggest that in T cells, α7-nAChR, despite its close sequence homology with neuronal α7-nAChR, fails to form a ligand-gated Ca2+ channel, and that the nicotine-induced rise in [Ca2+]i in T cells requires functional TCR/CD3 and leukocyte-specific tyrosine kinase.

Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes.

BackgroundMeiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., Saccharomyces cerevisiae and Drosophila melanogaster), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.ResultsA high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.ConclusionThese findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.

Immunosuppressive and Anti-Inflammatory Effects of Nicotine Administered by Patch in an Animal Model

ABSTRACT To study the immunological effects of nicotine, there are several rodent models for chronic nicotine administration. These models include subcutaneously implanted miniosmotic pumps, nicotine-spiked drinking water, and self-administration via jugular cannulae. Administration of nicotine via these routes affects the immune system. Smokers frequently use nicotine patches to quit smoking, and the immunological effects of nicotine patches are largely unknown. To determine whether the nicotine patch affects the immune system, nicotine patches were affixed daily onto the backs of Lewis rats for 3 to 4 weeks. The patches efficiently raised the levels of nicotine and cotinine in serum and strongly inhibited the antibody-forming cell response of spleen cells to sheep red blood cells. The nicotine patch also suppressed the concanavalin A-induced T-cell proliferation and mobilization of intracellular Ca2+ by spleen cells, as well as the fever response of animals to subcutaneous administration of turpentine. Moreover, immunosuppression was associated with chronic activation of protein tyrosine kinase and phospholipase C-γ1 activities. Thus, in this animal model of nicotine administration, the nicotine patch efficiently raises the levels of nicotine and cotinine in serum and impairs both the immune and inflammatory responses.

Host gene expression classifiers diagnose acute respiratory illness etiology.

Pathogen-specific host gene expression changes may combat inappropriate antibiotic use and emerging antibiotic resistance. Resisting antibiotics No matter the cause, acute respiratory infections can be miserable. Indeed, these infections are one of the most common reasons for seeking medical care. A clear diagnostic can help medical practitioners resist the patient-induced pressure to prescribe antibiotics as a catch-all therapy, which increases the risk of bacteria developing antibiotic resistance. Now, Tsalik et al. report clear differences in host gene expression induced by bacterial and viral infection as well as by noninfectious illness. These differences can be used to discriminate between these groups, and a host gene expression classifier may be a helpful diagnostic platform to curb unnecessary antibiotic use. Acute respiratory infections caused by bacterial or viral pathogens are among the most common reasons for seeking medical care. Despite improvements in pathogen-based diagnostics, most patients receive inappropriate antibiotics. Host response biomarkers offer an alternative diagnostic approach to direct antimicrobial use. This observational cohort study determined whether host gene expression patterns discriminate noninfectious from infectious illness and bacterial from viral causes of acute respiratory infection in the acute care setting. Peripheral whole blood gene expression from 273 subjects with community-onset acute respiratory infection (ARI) or noninfectious illness, as well as 44 healthy controls, was measured using microarrays. Sparse logistic regression was used to develop classifiers for bacterial ARI (71 probes), viral ARI (33 probes), or a noninfectious cause of illness (26 probes). Overall accuracy was 87% (238 of 273 concordant with clinical adjudication), which was more accurate than procalcitonin (78%, P < 0.03) and three published classifiers of bacterial versus viral infection (78 to 83%). The classifiers developed here externally validated in five publicly available data sets (AUC, 0.90 to 0.99). A sixth publicly available data set included 25 patients with co-identification of bacterial and viral pathogens. Applying the ARI classifiers defined four distinct groups: a host response to bacterial ARI, viral ARI, coinfection, and neither a bacterial nor a viral response. These findings create an opportunity to develop and use host gene expression classifiers as diagnostic platforms to combat inappropriate antibiotic use and emerging antibiotic resistance.

Nicotine Primarily Suppresses Lung Th2 but Not Goblet Cell and Muscle Cell Responses to Allergens

Allergic asthma, an inflammatory disease characterized by the infiltration and activation of various leukocytes, the production of Th2 cytokines and leukotrienes, and atopy, also affects the function of other cell types, causing goblet cell hyperplasia/hypertrophy, increased mucus production/secretion, and airway hyperreactivity. Eosinophilic inflammation is a characteristic feature of human asthma, and recent evidence suggests that eosinophils also play a critical role in T cell trafficking in animal models of asthma. Nicotine is an anti-inflammatory, but the association between smoking and asthma is highly contentious and some report that smoking cessation increases the risk of asthma in ex-smokers. To ascertain the effects of nicotine on allergy/asthma, Brown Norway rats were treated with nicotine and sensitized and challenged with allergens. The results unequivocally show that, even after multiple allergen sensitizations, nicotine dramatically suppresses inflammatory/allergic parameters in the lung including the following: eosinophilic/lymphocytic emigration; mRNA and/or protein expression of the Th2 cytokines/chemokines IL-4, IL-5, IL-13, IL-25, and eotaxin; leukotriene C4; and total as well as allergen-specific IgE. Although nicotine did not significantly affect hexosaminidase release, IgG, or methacholine-induced airway resistance, it significantly decreased mucus content in bronchoalveolar lavage; interestingly, however, despite the strong suppression of IL-4/IL-13, nicotine significantly increased the intraepithelial-stored mucosubstances and Muc5ac mRNA expression. These results suggest that nicotine modulates allergy/asthma primarily by suppressing eosinophil trafficking and suppressing Th2 cytokine/chemokine responses without reducing goblet cell metaplasia or mucous production and may explain the lower risk of allergic diseases in smokers. To our knowledge this is the first direct evidence that nicotine modulates allergic responses.

Metabolomic derangements are associated with mortality in critically ill adult patients.

Objective To identify metabolomic biomarkers predictive of Intensive Care Unit (ICU) mortality in adults. Rationale Comprehensive metabolomic profiling of plasma at ICU admission to identify biomarkers associated with mortality has recently become feasible. Methods We performed metabolomic profiling of plasma from 90 ICU subjects enrolled in the BWH Registry of Critical Illness (RoCI). We tested individual metabolites and a Bayesian Network of metabolites for association with 28-day mortality, using logistic regression in R, and the CGBayesNets Package in MATLAB. Both individual metabolites and the network were tested for replication in an independent cohort of 149 adults enrolled in the Community Acquired Pneumonia and Sepsis Outcome Diagnostics (CAPSOD) study. Results We tested variable metabolites for association with 28-day mortality. In RoCI, nearly one third of metabolites differed among ICU survivors versus those who died by day 28 (N = 57 metabolites, p<.05). Associations with 28-day mortality replicated for 31 of these metabolites (with p<.05) in the CAPSOD population. Replicating metabolites included lipids (N = 14), amino acids or amino acid breakdown products (N = 12), carbohydrates (N = 1), nucleotides (N = 3), and 1 peptide. Among 31 replicated metabolites, 25 were higher in subjects who progressed to die; all 6 metabolites that are lower in those who die are lipids. We used Bayesian modeling to form a metabolomic network of 7 metabolites associated with death (gamma-glutamylphenylalanine, gamma-glutamyltyrosine, 1-arachidonoylGPC(20:4), taurochenodeoxycholate, 3-(4-hydroxyphenyl) lactate, sucrose, kynurenine). This network achieved a 91% AUC predicting 28-day mortality in RoCI, and 74% of the AUC in CAPSOD (p<.001 in both populations). Conclusion Both individual metabolites and a metabolomic network were associated with 28-day mortality in two independent cohorts. Metabolomic profiling represents a valuable new approach for identifying novel biomarkers in critically ill patients.

Nicotine Inhibits FcεRI-Induced Cysteinyl Leukotrienes and Cytokine Production without Affecting Mast Cell Degranulation Through α7/α9/α10-Nicotinic Receptors

Smokers are less likely to develop some inflammatory and allergic diseases. In Brown-Norway rats, nicotine inhibits several parameters of allergic asthma, including the production of Th2 cytokines and the cysteinyl leukotriene LTC4. Cysteinyl leukotrienes are primarily produced by mast cells, and these cells play a central role in allergic asthma. Mast cells express a high-affinity receptor for IgE (FcεRI). Following its cross-linking, cells degranulate and release preformed inflammatory mediators (early phase) and synthesize and secrete cytokines/chemokines and leukotrienes (late phase). The mechanism by which nicotine modulates mast cell activation is unclear. Using α-bungarotoxin binding and quantitative PCR and PCR product sequencing, we showed that the rat mast/basophil cell line RBL-2H3 expresses nicotinic acetylcholine receptors (nAChRs) α7, α9, and α10; exposure to exceedingly low concentrations of nicotine (nanomolar), but not the biologically inactive metabolite cotinine, for ≥8 h suppressed the late phase (leukotriene/cytokine production) but not degranulation (histamine and hexosaminidase release). These effects were unrelated to those of nicotine on intracellular free calcium concentration but were causally associated with the inhibition of cytosolic phospholipase A2 activity and the PI3K/ERK/NF-κB pathway, including phosphorylation of Akt and ERK and nuclear translocation of NF-κB. The suppressive effect of nicotine on the late-phase response was blocked by the α7/α9-nAChR antagonists methyllycaconitine and α-bungarotoxin, as well as by small interfering RNA knockdown of α7-, α9-, or α10-nAChRs, suggesting a functional interaction between α7-, α9-, and α10-nAChRs that might explain the response of RBL cells to nanomolar concentrations of nicotine. This “hybrid” receptor might serve as a target for novel antiallergic/antiasthmatic therapies.

Prenatal Secondhand Cigarette Smoke Promotes Th2 Polarization and Impairs Goblet Cell Differentiation and Airway Mucus Formation

Parental, particularly maternal, smoking increases the risk for childhood allergic asthma and infection. Similarly, in a murine allergic asthma model, prenatal plus early postnatal exposure to secondhand cigarette smoke (SS) exacerbates airways hyperreactivity and Th2 responses in the lung. However, the mechanism and contribution of prenatal versus early postnatal SS exposure on allergic asthma remain unresolved. To identify the effects of prenatal and/or early postnatal SS on allergic asthma, BALB/c dams and their offspring were exposed gestationally and/or 8–10 wk postbirth to filtered air or SS. Prenatal, but not postnatal, SS strongly increased methacholine and allergen (Aspergillus)-induced airway resistance, Th2 cytokine levels, and atopy and activated the Th2-polarizing pathway GATA3/Lck/ERK1/2/STAT6. Either prenatal and/or early postnatal SS downregulated the Th1-specific transcription factor T-bet and, surprisingly, despite high levels of IL-4/IL-13, dramatically blocked the allergen-induced mucous cell metaplasia, airway mucus formation, and the expression of mucus-related genes/proteins: Muc5ac, γ-aminobutyric acid A receptors, and SAM pointed domain-containing Ets-like factor. Given that SS/nicotine exposure of normal adult mice promotes mucus formation, the results suggested that fetal and neonatal lung are highly sensitive to cigarette smoke. Thus, although the gestational SS promotes Th2 polarization/allergic asthma, it may also impair and/or delay the development of fetal and neonatal lung, affecting mucociliary clearance and Th1 responses. Together, this may explain the increased susceptibility of children from smoking parents to allergic asthma and childhood respiratory infections.