A public resource facilitating clinical use of genomes.

Rapid advances in DNA sequencing promise to enable new diagnostics and individualized therapies. Achieving personalized medicine, however, will require extensive research on highly reidentifiable, integrated datasets of genomic and health information. To assist with this, participants in the Personal Genome Project choose to forgo privacy via our institutional review board- approved "open consent" process. The contribution of public data and samples facilitates both scientific discovery and standardization of methods. We present our findings after enrollment of more than 1,800 participants, including whole-genome sequencing of 10 pilot participant genomes (the PGP-10). We introduce the Genome-Environment-Trait Evidence (GET-Evidence) system. This tool automatically processes genomes and prioritizes both published and novel variants for interpretation. In the process of reviewing the presumed healthy PGP-10 genomes, we find numerous literature references implying serious disease. Although it is sometimes impossible to rule out a late-onset effect, stringent evidence requirements can address the high rate of incidental findings. To that end we develop a peer production system for recording and organizing variant evaluations according to standard evidence guidelines, creating a public forum for reaching consensus on interpretation of clinically relevant variants. Genome analysis becomes a two-step process: using a prioritized list to record variant evaluations, then automatically sorting reviewed variants using these annotations. Genome data, health and trait information, participant samples, and variant interpretations are all shared in the public domain-we invite others to review our results using our participant samples and contribute to our interpretations. We offer our public resource and methods to further personalized medical research.

Ethnicity and parental report of postoperative behavioral changes in children.

OBJECTIVES: To examine the role of ethnicity and language in parent report of children's postoperative behavioral recovery. AIM: To compare incidence of new onset negative behavior change in English- and Spanish-speaking White and Hispanic children following outpatient surgery. BACKGROUND: Postoperative behavioral change in children is common; however, it is unknown whether cultural variables including ethnicity and language may influence parent report of children's behavioral recovery. METHODS/MATERIALS: Participants included 288 parents (English-speaking White, English-speaking Hispanic, Spanish-speaking Hispanic parents) of children undergoing outpatient elective surgery. Parents completed the post-hospitalization behavior questionnaire (PHBQ) and parents' postoperative pain measure (PPPM) on postoperative days one, three, and seven at home. RESULTS: Most parents (83%) reported onset of new negative behavioral change in children postoperatively. Generalized estimating equations revealed significant group differences in overall behavior change [Wald χ(2)(12) = 375.69, P < 0.0001] after controlling for demographic and socioeconomic differences. At all three postoperative days, Spanish-speaking Hispanic (SSH) parents reported lower negative behavioral changes in their children compared to English-speaking White (ESW) parents (day 1: P < 0.01; day 3: P < 0.001; day 7: P < 0.10). On postoperative days one and three, SSH parents also reported lower total PHBQ scores than English-speaking Hispanic (ESH) parents [day 1: χ(2)(1) = 6.72, P = 0.01; day 3: χ(2)(1) = 7.98, P = 0.005]. CONCLUSION: The present study provides evidence that parent report of children's postoperative behavioral recovery may be influenced by cultural variables, such as ethnicity and language. The present results contribute to a growing body of evidence that highlights the need for culturally sensitive assessment and care of families in the medical setting. The findings may reflect differences in cultural values such as stoicism; however, future studies would benefit from examination of the factors that may account for the differences in reported behavior change after surgery (i.e., report bias, cultural values).

Multiplex padlock targeted sequencing reveals human hypermutable CpG variations.

Utilizing the full power of next-generation sequencing often requires the ability to perform large-scale multiplex enrichment of many specific genomic loci in multiple samples. Several technologies have been recently developed but await substantial improvements. We report the 10,000-fold improvement of a previously developed padlock-based approach, and apply the assay to identifying genetic variations in hypermutable CpG regions across human chromosome 21. From approximately 3 million reads derived from a single Illumina Genome Analyzer lane, approximately 94% (approximately 50,500) target sites can be observed with at least one read. The uniformity of coverage was also greatly improved; up to 93% and 57% of all targets fell within a 100- and 10-fold coverage range, respectively. Alleles at >400,000 target base positions were determined across six subjects and examined for single nucleotide polymorphisms (SNPs), and the concordance with independently obtained genotypes was 98.4%-100%. We detected >500 SNPs not currently in dbSNP, 362 of which were in targeted CpG locations. Transitions in CpG sites were at least 13.7 times more abundant than non-CpG transitions. Fractions of polymorphic CpG sites are lower in CpG-rich regions and show higher correlation with human-chimpanzee divergence within CpG versus non-CpG sites. This is consistent with the hypothesis that methylation rate heterogeneity along chromosomes contributes to mutation rate variation in humans. Our success suggests that targeted CpG resequencing is an efficient way to identify common and rare genetic variations. In addition, the significantly improved padlock capture technology can be readily applied to other projects that require multiplex sample preparation.

Clinical assessment incorporating a personal genome.

BACKGROUND: The cost of genomic information has fallen steeply, but the clinical translation of genetic risk estimates remains unclear. We aimed to undertake an integrated analysis of a complete human genome in a clinical context. METHODS: We assessed a patient with a family history of vascular disease and early sudden death. Clinical assessment included analysis of this patient's full genome sequence, risk prediction for coronary artery disease, screening for causes of sudden cardiac death, and genetic counselling. Genetic analysis included the development of novel methods for the integration of whole genome and clinical risk. Disease and risk analysis focused on prediction of genetic risk of variants associated with mendelian disease, recognised drug responses, and pathogenicity for novel variants. We queried disease-specific mutation databases and pharmacogenomics databases to identify genes and mutations with known associations with disease and drug response. We estimated post-test probabilities of disease by applying likelihood ratios derived from integration of multiple common variants to age-appropriate and sex-appropriate pre-test probabilities. We also accounted for gene-environment interactions and conditionally dependent risks. FINDINGS: Analysis of 2.6 million single nucleotide polymorphisms and 752 copy number variations showed increased genetic risk for myocardial infarction, type 2 diabetes, and some cancers. We discovered rare variants in three genes that are clinically associated with sudden cardiac death-TMEM43, DSP, and MYBPC3. A variant in LPA was consistent with a family history of coronary artery disease. The patient had a heterozygous null mutation in CYP2C19 suggesting probable clopidogrel resistance, several variants associated with a positive response to lipid-lowering therapy, and variants in CYP4F2 and VKORC1 that suggest he might have a low initial dosing requirement for warfarin. Many variants of uncertain importance were reported. INTERPRETATION: Although challenges remain, our results suggest that whole-genome sequencing can yield useful and clinically relevant information for individual patients. FUNDING: National Institute of General Medical Sciences; National Heart, Lung And Blood Institute; National Human Genome Research Institute; Howard Hughes Medical Institute; National Library of Medicine, Lucile Packard Foundation for Children's Health; Hewlett Packard Foundation; Breetwor Family Foundation.

Beyond pain: predictors of postoperative maladaptive behavior change in children.

UNLABELLED: OBJECTIVES & AIM: Using well-validated measures and controlling for potential confounding variables such as pain and surgical and anesthetic technique, the goal of this project was to identify the incidence of and risk factors for the development of behavior change in children after surgery. BACKGROUND: Although researchers have described maladaptive behavior change following surgery, many previous studies are limited by potential confounding variables, including postoperative pain, type of surgery, and surgical and anesthetic procedure. METHODS: Participants included 260 children undergoing tonsillectomy and adenoidectomy. Baseline and demographic data were collected prior to surgery and pain and behavioral recovery were recorded for 2 weeks following surgery. A standardized approach to anesthesia and surgical procedure was implemented and well-validated assessment measures were used. RESULTS: On the first day at home following surgery, 80.4% of children exhibited negative behavior change. Nearly one-third of children continued to exhibit behavior changes 2 weeks after surgery. Logistic regression analyses that controlled for pain severity identified several predictors of behavior change: preexisting somatic and anxious/depressed problems predicted new onset postoperative general anxiety, chi(2) (8) = 20.10, P = 0.010; younger age predicted separation anxiety, chi(2) (4) = 20.41, P < 0.01; and inhibited temperament predicted postoperative sleep disturbance, chi(2) (2) = 9.19, P = 0.010. CONCLUSIONS: Individual child factors above and beyond pain predict maladaptive postoperative behavior change; identification of these predictors may be helpful in both preventing and ameliorating difficulties with behavioral recovery following surgery.

Preoperative melatonin and its effects on induction and emergence in children undergoing anesthesia and surgery.

BACKGROUND: Studies conducted in adults undergoing surgery reported a beneficial effect of oral melatonin administered before surgery. There is a paucity of such data in children undergoing anesthesia and surgery. METHODS: Children undergoing surgery were randomly assigned to receive preoperatively oral midazolam 0.5 mg/kg or oral melatonin 0.05 mg/kg, 0.2 mg/kg, or 0.4 mg/kg. The primary outcome of the study was preoperative anxiety (Yale Preoperative Anxiety Scale). The secondary outcomes were the children's compliance with induction (Induction Compliance Checklist), emergence behavior (Keegan scale), and parental anxiety (State-Trait Anxiety Inventory). RESULTS: Repeated measures ANOVA showed that children who received melatonin at any of the three doses were more anxious compared with children who received midazolam (P < 0.001). Parental anxiety did not differ on the basis of the experimental condition (P = ns). The melatonin groups showed a dose-response effect on emergence behavior. Children who received melatonin developed less emergence delirium compared with those who received midazolam (P < 0.05), and the effect was dose related; the incidence after 0.05 mg/kg melatonin was 25.0%, incidence after 0.2 mg/kg melatonin was 8.3%, and incidence after 0.4 mg/kg melatonin was 5.4%. CONCLUSIONS: Midazolam is more effective than melatonin in reducing children's anxiety at induction of anesthesia. Melatonin showed a direct dose-dependent effect on emergence delirium.

The place of premedication in pediatric practice.

Behind the multiple arguments for and against the use of premedication, sedative drugs in children is a noble principle that of minimizing psychological trauma related to anesthesia and surgery. However, several confounding factors make it very difficult to reach didactic evidence-based conclusions. One of the key confounding issues is that the nature of expectations and responses for both parent and child vary greatly in different environments around the world. Studies applicable to one culture and to one hospital system (albeit multicultural) may not apply elsewhere. Moreover, the study of hospital-related distress begins at the start of the patient's journey and ends long after hospital discharge; it cannot be focused completely on just the moment of anesthetic induction. Taking an example from actual practice experience, the trauma caused by the actual giving of a premedication to a child who absolutely does not want it and may struggle may not be recorded in a study but could form a significant component of overall effect and later psychological pathology. Clearly, attitudes by health professionals and parents to the practice of routine pediatric premedication, vary considerably, often provoking strong opinions. In this pro-con article we highlight two very different approaches to premedication. It is hoped that this helps the reader to critically re-evaluate a practice, which was universal historically and now in many centers is more selective.

Novel portable device measures preoperative patient metabolic gas exchange.

BACKGROUND: Indirect calorimetry (IC), the measurement of airway CO2 elimination (VCO2), O2 [corrected] uptake (VO2) [corrected], and respiratory exchange ratio (RER = VCO2/VO2), is a noninvasive modality for the assessment of body metabolism. In anesthesia, IC can signal critical events and onset of acute metabolic derangements. We have previously demonstrated the accuracy and precision of a new IC measurement system designed for mechanically ventilated patients, comprised of a new clinical bymixer, fast response humidity and temperature sensor, and a flowmeter. However, measurement of IC during spontaneous breathing is challenging because of unstable tidal volume, frequency, and functional residual capacity (FRC). METHODS: A new device for IC measurements, designed specifically for spontaneous breathing, was validated against a metabolic lung simulator bench setup. In a second study, the same device was used to conduct preoperative measurements of VCO2 and VO2 in 15 patients. RESULTS: Our measurements showed excellent correlation and agreement with metabolic lung simulator values: The average (+/-SD) percent error for airway VCO2 was -4.7% +/- 3.31%; the average (+/-SD) percent error for airway VO2 was -0.30% +/- 5.25%. Average values of VCO2 and VO2 in the patient study (3.01 +/- 0.56 and 3.44 +/- 0.69 mL x kg(-1) x min(-1), respectively) were in agreement with previously reported values. CONCLUSION: We have shown that the new, portable bymixer-flow device, using a bymixer and a fast response humidity sensor, provided accurate and convenient bedside measurement of VCO2 and VO2. We believe that it can contribute in the future to preoperative assessment and baseline reference value for perioperative management.

Human genome sequencing using unchained base reads on self-assembling DNA nanoarrays.

Genome sequencing of large numbers of individuals promises to advance the understanding, treatment, and prevention of human diseases, among other applications. We describe a genome sequencing platform that achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs. We sequenced three human genomes with this platform, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome. Validation of one genome data set demonstrates a sequence accuracy of about 1 false variant per 100 kilobases. The high accuracy, affordable cost of $4400 for sequencing consumables, and scalability of this platform enable complete human genome sequencing for the detection of rare variants in large-scale genetic studies.

Importance and interpretation of fast-response airway hygrometry during ventilation of anesthetized patients.

BACKGROUND: Measurement of oxygen uptake (Vo2) should help detect non-steady state critical events and metabolic derangement during anesthesia. Vo2 requires measurement of respiratory relative humidity (RH) and temperature (T). We have developed a fast response T and humidity sensor (HS), which uses tiny wet and dry thermometers to determine RH by psychrometry, where low RH causes evaporation to decrease wet T below dry T. In laboratory bench studies, we determined that >/=5 l/min gas flow through the HS is required for valid psychrometry function. This study demonstrates that monitoring of flow through the HS enhances the accuracy of RH measurement and interpretation. METHODS: Phase One: Laboratory bench validation; We designed a special bench setup for the validation of metabolic gas exchange compared to precise ethanol combustion. Phase 2: Clinical study; During mechanical ventilation of 6 anesthetized surgical patients, airway flow was used to successfully select valid wet T and dry T during inspiration and expiration, from which respective RH's were calculated using principles of psychrometry. RESULTS: The average (+/-SD) percent error for airway Vco2 (compared to the stoichiometric value) was -1.84 +/- 2.69% (Table 2). The average (+/-SD) percent error for airway Vo2 was 0.91 +/- 3.10%. Average RQ was 0.649 +/- 0.017. For all patients, average inspired RH was 36.1 +/- 11.8% (range of 17-52%), which differed significantly from expiration (103 +/- 9%). Among the 6-8 consecutive breaths for each patient, average standard deviations of expired RH were only 0.6%. CONCLUSION: We conclude that airway flow monitoring enhances the interpretation and accuracy of the fast-response HS measurements during inspiration and expiration, allowing for the determination of Vo2 in patients during anesthesia.

New metabolic lung simulator: development, description, and validation.

OBJECTIVE: Indirect calorimetry, the determination of airway carbon dioxide elimination (V(CO2),and oxygen uptake (V(O2)), can be used to non-invasively detect non-steady state perturbations of gas kinetics and mirror tissue metabolism. Validation of monitoring instruments in patients is difficult because there is no standard reference measurement, a wide range of physiologic values is required, and steady state is difficult to achieve and confirm. We present the development, critical details, and validation of a practical bench setup of a metabolic lung simulator, to generate a wide range of accurate, adjustable, and stable reference values of V(CO2) and V(O2), for development, calibration, and validation of indirect calorimetry methodology and clinical monitors. METHODS: We utilized a metered alcohol combustion system, which allowed safe, precise, and adjustable delivery of ethanol to a specially designed wick system to stoichiometrically generate reference V(CO2) and V(O2). Gas was pumped through a circular circuit between the separate metabolic chamber and mechanical lung, to preserve basic features of mammalian gas kinetics, including a physiologic ventilation waveform and the ability to induce non-steady state changes. Accurate and precise generation of V(CO2) and V(O2) were validated against separate measurements of gas flow and gas fractions in a collection bag. RESULTS: For volume control ventilation, average error for V(CO2) and V(O2) was -0.16% +/- 1.77 and 1.68% +/- 3.95, respectively. For pressure control ventilation, average error for V(CO2) and V(O2) was 0.90% +/- 2.48% and 4.86% +/- 2.21% respectively. Low values of measured ethanol vapor and carbon monoxide supported complete and pure combustion. CONCLUSIONS: The comprehensive description details the solutions to many problems, to help future investigations of metabolic gas exchange and contribute to improved patient monitoring during anesthesia and critical care medicine.

Bymixer system can measure O2 uptake and CO2 elimination in the anesthesia circle circuit.

BACKGROUND: The ability to measure carbon dioxide elimination (Vco(2)), oxygen uptake (Vo(2)), and R (respiratory exchange ratio, Vco(2)/Vo(2)) during anesthesia may help the non-invasive detection of critical events (e.g., abrupt decrease in cardiac output) and metabolic upset (e.g., onset of anaerobic metabolism). METHODS: We have developed a new clinical bymixer (inline mixing chamber) that can measure mixed inspired and expired gas fractions in the anesthesia circle circuit. The addition of a standard anesthesia gas analyzer and flowmeter, and a new airway temperature and humidity sensor, allow determinations of Vco(2) and Vo(2) at the airway opening of the circle circuit. Over a range of tidal volume and frequency, Vco(2) and Vo(2) were compared to reference values generated by the combustion of metered liquid ethanol in a new metabolic lung simulator. RESULTS: By linear regression, bymixer-flow measurements of Vco(2) (slope = 1.02, Y-intercept = -5.31, coefficient of determination, R(2) = 0.998) and Vo(2) (slope = 1.05, Y-intercept = -4.34, R(2) = 0.993) correlated closely to the reference values generated by the metabolic lung simulator. Limits of agreement analysis generated percent errors (mean +/- 1.96 SD) of -1.2 +/- 7.2% for Vco(2) and 2.5 +/- 9.8% for Vo(2). CONCLUSIONS: The new clinical bymixer is compact, lightweight, disposable, inexpensive, and has a fast and adjustable response time (time constant about 14 sec). Anesthesia circle circuit integrity is maintained. Bymixer-flow measurements of Vco(2) and Vo(2) are accurate and may add to clinical monitoring under anesthesia and surgery.

Accurate multiplex polony sequencing of an evolved bacterial genome.

We describe a DNA sequencing technology in which a commonly available, inexpensive epifluorescence microscope is converted to rapid nonelectrophoretic DNA sequencing automation. We apply this technology to resequence an evolved strain of Escherichia coli at less than one error per million consensus bases. A cell-free, mate-paired library provided single DNA molecules that were amplified in parallel to 1-micrometer beads by emulsion polymerase chain reaction. Millions of beads were immobilized in a polyacrylamide gel and subjected to automated cycles of sequencing by ligation and four-color imaging. Cost per base was roughly one-ninth as much as that of conventional sequencing. Our protocols were implemented with off-the-shelf instrumentation and reagents.

Novel, adjustable, clinical bymixer measures mixed expired gas concentrations in anesthesia circle circuit.

UNLABELLED: We have introduced a novel, parallel design into a new clinical bymixer (patent pending), named for the bypass of a constant fraction of total flow through a mixing chamber. Over a wide range of tidal volumes (300-1200 mL), frequency (6-20 breaths/min), and PCO(2) (6-50 mm Hg), the bymixer provided accurate measurement of mixed expired gas fractions in the ventilation circuit compared with an expired gas collection in a metabolic lung bench setup (average slope, 1.00; average y intercept, -0.01; average coefficient of determination, R(2) = 0.9988). Simple changes in mixing chamber volume provided adjustable bymixer response times. The fast bymixer response (time constant, 6.4 s) should allow measurements to be updated every 20 s (where 95% response occurs by three time constants). The new clinical bymixer is constructed from standard anesthesia circuit components, attaches easily to the anesthesia machine inspired outlet and expired inlet ports, is simple to clean and sterilize, and has no reservoir to trap condensed water vapor from expired gas. The new clinical bymixer may facilitate indirect calorimetry (CO(2) elimination, VCO(2), and oxygen uptake, VCO(2)) during anesthesia and the noninvasive detection of metabolic upset (e.g., onset of anaerobic metabolism) and critical events (e.g., pulmonary embolism). IMPLICATIONS: A new clinical bymixer (inline mixing chamber) provides a fast response and accurate measurements of mixed expired gas fractions in the anesthesia circle circuit. A novel parallel design facilitates adjustable response, easy cleaning, and construction from standard airway circuit components. The new clinical bymixer may facilitate widespread introduction of indirect calorimetry during anesthesia.

Measurement of oxygen uptake and carbon dioxide elimination using the bymixer: validation in a metabolic lung simulator.

BACKGROUND: The authors have developed a new clinical bymixer that bypasses a constant fraction of gas flow through a mixing arm. A separate bymixer was interposed in the expiratory and inspiratory limbs of the ventilation circuit to measure mixed gas fractions. By utilizing nitrogen conservation, the clinical bymixer allows the determination of airway carbon dioxide elimination (VCO2) and oxygen uptake (VO2), whenever basic expired flow and gas monitoring measurements are used for the patient. Neither an expiratory exhaust gas collection bag nor expensive, complex equipment are needed. This study tested the accuracy of airway bymixer-flow measurements of VCO2 and VO2 in a new bench apparatus. METHODS: The authors compared airway bymixer-flow measurements of VCO2 and VO2 over a range of reference values generated by ethanol combustion in a new metabolic lung simulator, which was ventilated by a volume-cycled respirator. An airway humidity and temperature sensor permitted standard temperature and pressure, dry, correction of airway VCO2 and VO2. RESULTS: Bymixer-flow airway measurements of VCO2 and VO2 correlated closely (R2 = 0.999 and 0.998, respectively) with the stoichiometric values generated by ethanol combustion. Limits of agreement for VCO2 and VO2 were 0.1 +/- 4.7 and 1.1 +/- 5.7%, respectively. The average (+/- SD) percent error for airway VCO2 (compared with the stoichiometric value) was 0.1 +/- 2.4%. The same error for airway VO2 was 1.1 +/- 2.9%. CONCLUSIONS: The new clinical bymixer, plus basic expired flow and gas fraction measurements, generated clinically accurate determinations of VCO2 and VO2. These measurements are helpful in the assessment of metabolic gas exchange in the critical care unit. In contrast to using the gas collection bag or complex metabolic monitor, the bymixer should measure mixed gas concentrations in the inspired or expired limb of the common anesthesia circle ventilation circuit.