Design Considerations for Massively Parallel Sequencing Studies of Complex Human Disease

Massively Parallel Sequencing (MPS) allows sequencing of entire exomes and genomes to now be done at reasonable cost, and its utility for identifying genes responsible for rare Mendelian disorders has been demonstrated. However, for a complex disease, study designs need to accommodate substantial degrees of locus, allelic, and phenotypic heterogeneity, as well as complex relationships between genotype and phenotype. Such considerations include careful selection of samples for sequencing and a well-developed strategy for identifying the few “true” disease susceptibility genes from among the many irrelevant genes that will be found to harbor rare variants. To examine these issues we have performed simulation-based analyses in order to compare several strategies for MPS sequencing in complex disease. Factors examined include genetic architecture, sample size, number and relationship of individuals selected for sequencing, and a variety of filters based on variant type, multiple observations of genes and concordance of genetic variants within pedigrees. A two-stage design was assumed where genes from the MPS analysis of high-risk families are evaluated in a secondary screening phase of a larger set of probands with more modest family histories. Designs were evaluated using a cost function that assumes the cost of sequencing the whole exome is 400 times that of sequencing a single candidate gene. Results indicate that while requiring variants to be identified in multiple pedigrees and/or in multiple individuals in the same pedigree are effective strategies for reducing false positives, there is a danger of over-filtering so that most true susceptibility genes are missed. In most cases, sequencing more than two individuals per pedigree results in reduced power without any benefit in terms of reduced overall cost. Further, our results suggest that although no single strategy is optimal, simulations can provide important guidelines for study design

From DNA sequence to application: possibilities and complications

The development of sophisticated genetic tools during the past 15 years have facilitated a tremendous increase of fundamental and application-oriented knowledge of lactic acid bacteria (LAB) and their bacteriophages. This knowledge relates both to the assignments of open reading frames (ORF’s) and the function of non-coding DNA sequences. Comparison of the complete nucleotide sequences of several LAB bacteriophages has revealed that their chromosomes have a fixed, modular structure, each module having a set of genes involved in a specific phase of the bacteriophage life cycle. LAB bacteriophage genes and DNA sequences have been used for the construction of temperature-inducible gene expression systems, gene-integration systems, and bacteriophage defence systems. The function of several LAB open reading frames and transcriptional units have been identified and characterized in detail. Many of these could find practical applications, such as induced lysis of LAB to enhance cheese ripening and re-routing of carbon fluxes for the production of a specific amino acid enantiomer. More knowledge has also become available concerning the function and structure of non-coding DNA positioned at or in the vicinity of promoters. In several cases the mRNA produced from this DNA contains a transcriptional terminator-antiterminator pair, in which the antiterminator can be stabilized either by uncharged tRNA or by interaction with a regulatory protein, thus preventing formation of the terminator so that mRNA elongation can proceed. Evidence has accumulated showing that also in LAB carbon catabolite repression in LAB is mediated by specific DNA elements in the vicinity of promoters governing the transcription of catabolic operons. Although some biological barriers have yet to be solved, the vast body of scientific information presently available allows the construction of tailor-made genetically modified LAB. Today, it appears that societal constraints rather than biological hurdles impede the use of genetically modified LAB.

Predicting Infectious ComplicatioNs in Children with Cancer : an external validation study

Background:The aim of this study was to validate the 'Predicting Infectious ComplicatioNs in Children with Cancer' (PICNICC) clinical decision rule (CDR) that predicts microbiologically documented infection (MDI) in children with cancer and fever and neutropenia (FN). We also investigated costs associated with current FN management strategies in Australia.Methods:Demographic, episode, outcome and cost data were retrospectively collected on 650 episodes of FN. We assessed the discrimination, calibration, sensitivity and specificity of the PICNICC CDR in our cohort compared with the derivation data set.Results:Using the original variable coefficients, the CDR performed poorly. After recalibration the PICNICC CDR had an area under the receiver operating characteristic (AUC-ROC) curve of 0.638 (95% CI 0.590-0.685) and calibration slope of 0.24. The sensitivity, specificity, positive predictive value and negative predictive value of the PICNICC CDR at presentation was 78.4%, 39.8%, 28.6% and 85.7%, respectively. For bacteraemia, the sensitivity improved to 85.2% and AUC-ROC to 0.71. Application at day 2, taking into consideration the proportion of MDI known (43%), further improved the sensitivity to 87.7%. Length of stay is the main contributor to cost of FN treatment, with an average cost per day of AUD 2183 in the low-risk group.Conclusions:For prediction of any MDI, the PICNICC rule did not perform as well at presentation in our cohort as compared with the derivation study. However, for bacteraemia, the predictive ability was similar to that of the derivation study, highlighting the importance of recalibration using local data. Performance also improved after an overnight period of observation. Implementation of a low-risk pathway, using the PICNICC CDR after a short period of inpatient observation, is likely to be safe and has the potential to reduce health-care expenditure

Statistical Guidance for Experimental Design and Data Analysis of Mutation Detection in Rare Monogenic Mendelian Diseases by Exome Sequencing

Recently, whole-genome sequencing, especially exome sequencing, has successfully led to the identification of causal mutations for rare monogenic Mendelian diseases. However, it is unclear whether this approach can be generalized and effectively applied to other Mendelian diseases with high locus heterogeneity. Moreover, the current exome sequencing approach has limitations such as false positive and false negative rates of mutation detection due to sequencing errors and other artifacts, but the impact of these limitations on experimental design has not been systematically analyzed. To address these questions, we present a statistical modeling framework to calculate the power, the probability of identifying truly disease-causing genes, under various inheritance models and experimental conditions, providing guidance for both proper experimental design and data analysis. Based on our model, we found that the exome sequencing approach is well-powered for mutation detection in recessive, but not dominant, Mendelian diseases with high locus heterogeneity. A disease gene responsible for as low as 5% of the disease population can be readily identified by sequencing just 200 unrelated patients. Based on these results, for identifying rare Mendelian disease genes, we propose that a viable approach is to combine, sequence, and analyze patients with the same disease together, leveraging the statistical framework presented in this work

Somatic PIK3R1 Variation as a Cause of Vascular Malformations and Overgrowth

PurposeSomatic activating variants in the PI3K-AKT pathway cause vascular malformations with and without overgrowth. We previously reported an individual with capillary and lymphatic malformation harboring a pathogenic somatic variant in PIK3R1, which encodes three PI3K complex regulatory subunits. Here, we investigate PIK3R1 in a large cohort with vascular anomalies and identify an additional 16 individuals with somatic mosaic variants in PIK3R1.MethodsAffected tissue from individuals with vascular lesions and overgrowth recruited from a multisite collaborative network was studied. Next-generation sequencing targeting coding regions of cell-signaling and cancer-associated genes was performed followed by assessment of variant pathogenicity.ResultsThe phenotypic and variant spectrum associated with somatic variation in PIK3R1 is reported herein. Variants occurred in the inter-SH2 or N-terminal SH2 domains of all three PIK3R1 protein products. Phenotypic features overlapped those of the PIK3CA-related overgrowth spectrum (PROS). These overlapping features included mixed vascular malformations, sandal toe gap deformity with macrodactyly, lymphatic malformations, venous ectasias, and overgrowth of soft tissue or bone.ConclusionSomatic PIK3R1 variants sharing attributes with cancer-associated variants cause complex vascular malformations and overgrowth. The PIK3R1-associated phenotypic spectrum overlaps with PROS. These data extend understanding of the diverse phenotypic spectrum attributable to genetic variation in the PI3K-AKT pathway

Gravitational waves from single neutron stars: an advanced detector era survey

With the doors beginning to swing open on the new gravitational wave astronomy, this review provides an up-to-date survey of the most important physical mechanisms that could lead to emission of potentially detectable gravitational radiation from isolated and accreting neutron stars. In particular we discuss the gravitational wave-driven instability and asteroseismology formalism of the f- and r-modes, the different ways that a neutron star could form and sustain a non-axisymmetric quadrupolar "mountain" deformation, the excitation of oscillations during magnetar flares and the possible gravitational wave signature of pulsar glitches. We focus on progress made in the recent years in each topic, make a fresh assessment of the gravitational wave detectability of each mechanism and, finally, highlight key problems and desiderata for future work.Comment: 39 pages, 12 figures, 2 tables. Chapter of the book "Physics and Astrophysics of Neutron Stars", NewCompStar COST Action 1304. Minor corrections to match published versio

The search for transient astrophysical neutrino emission with IceCube-DeepCore

We present the results of a search for astrophysical sources of brief transient neutrino emission using IceCube and DeepCore data acquired between 2012 May 15 and 2013 April 30. While the search methods employed in this analysis are similar to those used in previous IceCube point source searches, the data set being examined consists of a sample of predominantly sub-TeV muon-neutrinos from the Northern Sky (-5 degrees < delta < 90 degrees) obtained through a novel event selection method. This search represents a first attempt by IceCube to identify astrophysical neutrino sources in this relatively unexplored energy range. The reconstructed direction and time of arrival of neutrino events are used to search for any significant self-correlation in the data set. The data revealed no significant source of transient neutrino emission. This result has been used to construct limits at timescales ranging from roughly 1 s to 10 days for generic soft-spectra transients. We also present limits on a specific model of neutrino emission from soft jets in core-collapse supernovae

Altered Metabolic Signature in Pre-Diabetic NOD Mice

Altered metabolism proceeding seroconversion in children progressing to Type 1 diabetes has previously been demonstrated. We tested the hypothesis that non-obese diabetic (NOD) mice show a similarly altered metabolic profile compared to C57BL/6 mice. Blood samples from NOD and C57BL/6 female mice was collected at 0, 1, 2, 3, 4, 5, 6, 7, 9, 11, 13 and 15 weeks and the metabolite content was analyzed using GC-MS. Based on the data of 89 identified metabolites OPLS-DA analysis was employed to determine the most discriminative metabolites. In silico analysis of potential involved metabolic enzymes was performed using the dbSNP data base. Already at 0 weeks NOD mice displayed a unique metabolic signature compared to C57BL/6. A shift in the metabolism was observed for both strains the first weeks of life, a pattern that stabilized after 5 weeks of age. Multivariate analysis revealed the most discriminative metabolites, which included inosine and glutamic acid. In silico analysis of the genes in the involved metabolic pathways revealed several SNPs in either regulatory or coding regions, some in previously defined insulin dependent diabetes (Idd) regions. Our result shows that NOD mice display an altered metabolic profile that is partly resembling the previously observation made in children progressing to Type 1 diabetes. The level of glutamic acid was one of the most discriminative metabolites in addition to several metabolites in the TCA cycle and nucleic acid components. The in silico analysis indicated that the genes responsible for this reside within previously defined Idd regions

Stereotactic body radiotherapy for organ-confined prostate cancer

<p>Abstract</p> <p>Background</p> <p>Improved understanding of prostate cancer radiobiology combined with advances in delivery of radiation to the moving prostate offer the potential to reduce treatment-related morbidity and maintain quality of life (QOL) following prostate cancer treatment. We present preliminary results following stereotactic body radiotherapy (SBRT) treatment for organ-confined prostate cancer.</p> <p>Methods</p> <p>SBRT was performed on 304 patients with clinically localized prostate cancer: 50 received 5 fractions of 7 Gy (total dose 35 Gy) and 254 received 5 fractions of 7.25 Gy (total dose 36.25 Gy). Acute and late toxicity was assessed using the Radiation Therapy Oncology Group scale. The Expanded Prostate Cancer Index Composite questionnaire was used to assess QOL. Prostate-specific antigen response was monitored.</p> <p>Results</p> <p>At a median 30-month (26 - 37 month, range) follow-up there were no biochemical failures for the 35-Gy dose level. Acute Grade II urinary and rectal toxicities occurred in 4% of patients with no higher Grade acute toxicities. One Grade II late urinary toxicity occurred with no other Grade II or higher late toxicities. At a median 17-month (8 - 27 month, range) follow-up the 36.25 Gy dose level had 2 low- and 2 high-risk patients fail biochemically (biopsy showed 2 low- and 1 high-risk patients were disease-free in the gland). Acute Grade II urinary and rectal toxicities occurred in 4.7% (12/253) and 3.6% (9/253) of patients, respectively. For those patients with a minimum of 12 months follow-up, 5.8% (12/206) had late Grade II urinary toxicity and 2.9% (6/206) had late Grade II rectal toxicities. One late Grade III urinary toxicity occurred; no Grade IV toxicities occurred. For both dose levels at 17 months, bowel and urinary QOL returned to baseline values; sexual QOL decreased by 10%.</p> <p>Conclusions</p> <p>The low toxicity and maintained QOL are highly encouraging. Additional follow-up is needed to determine long-term biochemical control and maintenance of low toxicity and QOL.</p

The TNF-α antagonist etanerceptreverses age-related decreases in colonic SERT expression and faecal output in mice

Treatment for chronic constipation in older people is challenging and the condition has a major impact on quality of life. A lack of understanding about the causes of this condition has hampered the development of effective treatments. 5-HT is an important pro-kinetic agent in the colon. We examined whether alterations in colonic 5-HT signalling underlie age–related changes in faecal output in mice and whether these changes were due to an increase in TNF-α. Components of the 5-HT signalling system (5-HT, 5-HIAA, SERT) and TNF-α expression were examined in the distal colon of 3, 12, 18 and 24- month old mice and faecal output and water content monitored under control conditions and following the administration of etanercept (TNF-α inhibitor; 1 mg Kg-1). Faecal output and water content were reduced in aged animals. Age increased mucosal 5-HT availability and TNF-α expression and decreased mucosal SERT expression and 5-HIAA. Etanercept treatment of old mice reversed these changes, suggesting that age-related changes in TNFα expression are an important regulator of mucosal 5-HT signalling and pellet output and water content in old mice. These data point to “anti-TNFα” drugs as potential treatments for age-related chronic constipation