A three-stage approach for genome-wide association studies with family data for quantitative traits

Background: Genome-wide association (GWA) studies that use population-based association approaches may identify spurious associations in the presence of population admixture. In this paper, we propose a novel three-stage approach that is computationally efficient and robust to population admixture and more powerful than the family-based association test (FBAT) for GWA studies with family data. We propose a three-stage approach for GWA studies with family data. The first stage is to perform linear regression ignoring phenotypic correlations among family members. SNPs with a first stage p-value below a liberal cut-off (e.g. 0.1) are then analyzed in the second stage that employs a linear mixed effects (LME) model that accounts for within family correlations. Next, SNPs that reach genome-wide significance (e.g. $10^{-6}$ for 34,625 genotyped SNPs in this paper) are analyzed in the third stage using FBAT, with correction of multiple testing only for SNPs that enter the third stage. Simulations are performed to evaluate type I error and power of the proposed method compared to LME adjusting for 10 principal components (PC) of the genotype data. We also apply the three-stage approach to the GWA analyses of uric acid in Framingham Heart Study's SNP Health Association Resource (SHARe) project. Results: Our simulations show that whether or not population admixture is present, the three-stage approach has no inflated type I error. In terms of power, using LME adjusting PC is only slightly more powerful than the three-stage approach. When applied to the GWA analyses of uric acid in the SHARe project of FHS, the three-stage approach successfully identified and confirmed three SNPs previously reported as genome-wide significant signals. Conclusions: For GWA analyses of quantitative traits with family data, our three-stage approach provides another appealing solution to population admixture, in addition to LME adjusting for genetic PC

A Rapid and Economic In-House DNA Purification Method Using Glass Syringe Filters

Background Purity, yield, speed and cost are important considerations in plasmid purification, but it is difficult to achieve all of these at the same time. Currently, there are many protocols and kits for DNA purification, however none maximize all four considerations. Methodology/Principal Findings We now describe a fast, efficient and economic in-house protocol for plasmid preparation using glass syringe filters. Plasmid yield and quality as determined by enzyme digestion and transfection efficiency were equivalent to the expensive commercial kits. Importantly, the time required for purification was much less than that required using a commercial kit. Conclusions/Significance This method provides DNA yield and quality similar to that obtained with commercial kits, but is more rapid and less costly.This research was supported by Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewe

Hundreds of variants clustered in genomic loci and biological pathways affect human height

Most common human traits and diseases have a polygenic pattern of inheritance: DNA sequence variants at many genetic loci influence the phenotype. Genome-wide association (GWA) studies have identified more than 600 variants associated with human traits, but these typically explain small fractions of phenotypic variation, raising questions about the use of further studies. Here, using 183,727 individuals, we show that hundreds of genetic variants, in at least 180 loci, influence adult height, a highly heritable and classic polygenic trait. The large number of loci reveals patterns with important implications for genetic studies of common human diseases and traits. First, the 180 loci are not random, but instead are enriched for genes that are connected in biological pathways (P = 0.016) and that underlie skeletal growth defects (P < 0.001). Second, the likely causal gene is often located near the most strongly associated variant: in 13 of 21 loci containing a known skeletal growth gene, that gene was closest to the associated variant. Third, at least 19 loci have multiple independently associated variants, suggesting that allelic heterogeneity is a frequent feature of polygenic traits, that comprehensive explorations of already-discovered loci should discover additional variants and that an appreciable fraction of associated loci may have been identified. Fourth, associated variants are enriched for likely functional effects on genes, being over-represented among variants that alter amino-acid structure of proteins and expression levels of nearby genes. Our data explain approximately 10% of the phenotypic variation in height, and we estimate that unidentified common variants of similar effect sizes would increase this figure to approximately 16% of phenotypic variation (approximately 20% of heritable variation). Although additional approaches are needed to dissect the genetic architecture of polygenic human traits fully, our findings indicate that GWA studies can identify large numbers of loci that implicate biologically relevant genes and pathways.

Developing decision support tools incorporating personalised predictions of likely visual benefit versus harm for cataract surgery:research programme

Background Surgery for established cataract is highly cost-effective and uncontroversial, yet uncertainty remains for individuals about when to proceed and when to delay surgery during the earlier stages of cataract. Objective We aimed to improve decision-making for cataract surgery through the development of evidence-based clinical tools that provide general information and personalised risk/benefit information. Design We used a mixed methodology consisting of four work packages. Work package 1 involved the development and psychometric validation of a brief, patient self-reported measure of visual difficulty from cataract and its relief from surgery, named Cataract Patient-Reported Outcome Measure, five items (Cat-PROM5). Work package 2 involved the review and refinement of risk models for adverse surgical events (posterior capsule rupture and visual acuity loss related to cataract surgery). Work package 3 involved the development of prediction models for the Cat-PROM5-based self-reported outcomes from a cohort study of 1500 patients; assessment of the validity of preference-based health economic indices for cataract surgery and the calibration of these to Cat-PROM5; assessment of patients’ and health-care professionals’ views on risk–benefit presentation formats, the perceived usefulness of Cat-PROM5, the value of personalised risk–benefit information, high-value information items and shared decision-making; development of cataract decision aid frequently asked questions, incorporation of personalised estimates of risks and benefits; and development of a cataract decision quality measure to assess the quality of decision-making. Work package 4 involved a mixed-methods feasibility study for a fully powered randomised controlled trial of the use of the cataract decision aid and a qualitative study of discordant or mismatching perceptions of outcome between patients and health-care professionals. Setting Four English NHS recruitment centres were involved: Bristol (lead centre), Brighton, Gloucestershire and Torbay. Multicentre NHS cataract surgery data were obtained from the National Ophthalmology Database. Participants Work package 1 – participants (n = 822) were from all four centres. Work package 2 – electronic medical record data were taken from the National Ophthalmology Database (final set > 1M operations). Work package 3 – cohort study participants were from Bristol (n = 1200) and Gloucestershire (n = 300); qualitative and development work was undertaken with patients and health-care professionals from all four centres. Work package 4 – Bristol, Brighton and Torbay participated in the recruitment of patients (n = 42) for the feasibility trial and recruitment of health-care professionals for the qualitative elements. Interventions For the feasibility trial, the intervention was the use of the cataract decision aid, incorporating frequently asked questions and personalised estimations of both adverse outcomes and self-reported benefit. Main outcome measures There was a range of quantitative and qualitative outcome measures: questionnaire psychometric performance metrics, risk indicators of adverse surgical events and visual outcome, predictors of self-reported outcome following cataract surgery, patient and health-care practitioner views, health economic calibration measures and randomised controlled trial feasibility measures. Data sources The data sources were patient self-reported questionnaire responses, study clinical data collection forms, recorded interviews with patients and health-care professionals, and anonymised National Ophthalmology Database data. Results Work package 1 – Cat-PROM5 was developed and validated with excellent to good psychometric properties (Rasch reliability 0.9, intraclass correlation repeatability 0.9, unidimensionality with residual eigenvalues ≤ 1.5) and excellent responsiveness to surgical intervention (Cohen delta –1.45). Work package 2 – earlier risk models for posterior capsule rupture and visual acuity loss were broadly affirmed (C-statistic for posterior capsule rupture 0.64; visual acuity loss 0.71). Work package 3 – the Cat-PROM5-based self-reported outcome regression models were derived based on 1181 participants with complete data (R2 ≈ 30% for each). Of the four preference-based health economic indices assessed, two demonstrated reasonable performance. Cat-PROM5 was successfully calibrated to health economic indices; adjusted limited dependent variable mixture models offered good to excellent fit (root-mean-square error 0.10–0.16). The personalised quantitative risk information was generally perceived as beneficial. A cataract decision aid and cataract decision quality measure were successfully developed based on the views of patients and health-care professionals. Work package 4 – data completeness was good for the feasibility study primary and secondary variables both before and after intervention/surgery (data completeness range 100–88%). Considering ability to recruit, the sample size required, instrumentation and availability of necessary health economic data, a fully powered randomised controlled trial (patients, n = 800, effect size 0.2 standard deviations, power 80%; p = 0.05) of the cataract decision aid would be feasible following psychometric refinement of the primary outcome (the cataract decision quality measure). The cataract decision aid was generally well-received by patients and health-care professionals, with cautions raised regarding perceived time and workload barriers. Discordant outcomes mostly related to patient dissatisfaction, with no clinical problem found. Limitations The National Ophthalmology Database data are expected to include some errors (mitigated by large multicentre data aggregations). The feasibility randomised controlled trial primary outcome (the cataract decision quality measure) displayed psychometric imperfections requiring refinement. The clinical occurrence of discordant outcomes is uncommon and the study team experienced difficulty identifying patients in this situation. Future work Future work could include regular review of the risk models for adverse outcomes to ensure currency, and the technical precision of complex-numbers analysis of refractive outcome to invite opportunities to improve post-operative spectacle-free vision. In addition, a fully powered randomised controlled trial of the cataract decision aid would be feasible, following psychometric refinement of the primary outcome (the cataract decision quality measure); this would clarify its potential role in routine service delivery. Conclusions In this research programme, evidence-based clinical tools have been successfully developed to improve pre-operative decision-making in cataract surgery. These include a psychometrically robust, patient-reported outcome measure (Cat-PROM5); prediction models for patient self-reported outcomes using Cat-PROM5; prediction models for clinically adverse surgical events and adverse visual acuity outcomes; and a cataract decision aid with relevant general information and personalised risk/benefit predictions. In addition, the successful mapping of Cat-PROM5 to existing health economic indices was achieved and the performances of indices were assessed in patients undergoing cataract surgery. A future full-powered randomised controlled trial of the cataract decision aid would be feasible (patients, n = 800, effect size 0.2 standard deviations, power 80%; p = 0.05). Trial registration This trial is registered as ISRCTN11309852. Funding This project was funded by the National Institute for Health and Care Research (NIHR) Programme Grants for Applied Research programme and will be published in full in Programme Grants for Applied Research; Vol. 10, No. 9. See the NIHR Journals Library website for further project information

Framingham Heart Study 100K project: genome-wide associations for cardiovascular disease outcomes

BACKGROUND:Cardiovascular disease (CVD) and its most common manifestations - including coronary heart disease (CHD), stroke, heart failure (HF), and atrial fibrillation (AF) - are major causes of morbidity and mortality. In many industrialized countries, cardiovascular disease (CVD) claims more lives each year than any other disease. Heart disease and stroke are the first and third leading causes of death in the United States. Prior investigations have reported several single gene variants associated with CHD, stroke, HF, and AF. We report a community-based genome-wide association study of major CVD outcomes.METHODS:In 1345 Framingham Heart Study participants from the largest 310 pedigrees (54% women, mean age 33 years at entry), we analyzed associations of 70,987 qualifying SNPs (Affymetrix 100K GeneChip) to four major CVD outcomes: major atherosclerotic CVD (n = 142; myocardial infarction, stroke, CHD death), major CHD (n = 118; myocardial infarction, CHD death), AF (n = 151), and HF (n = 73). Participants free of the condition at entry were included in proportional hazards models. We analyzed model-based deviance residuals using generalized estimating equations to test associations between SNP genotypes and traits in additive genetic models restricted to autosomal SNPs with minor allele frequency [greater than or equal to]0.10, genotype call rate [greater than or equal to]0.80, and Hardy-Weinberg equilibrium p-value [greater than or equal to] 0.001.RESULTS:Six associations yielded p <10-5. The lowest p-values for each CVD trait were as follows: major CVD, rs499818, p = 6.6 x 10-6; major CHD, rs2549513, p = 9.7 x 10-6; AF, rs958546, p = 4.8 x 10-6; HF: rs740363, p = 8.8 x 10-6. Of note, we found associations of a 13 Kb region on chromosome 9p21 with major CVD (p 1.7 - 1.9 x 10-5) and major CHD (p 2.5 - 3.5 x 10-4) that confirm associations with CHD in two recently reported genome-wide association studies. Also, rs10501920 in CNTN5 was associated with AF (p = 9.4 x 10-6) and HF (p = 1.2 x 10-4). Complete results for these phenotypes can be found at the dbgap website http://www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007.CONCLUSION:No association attained genome-wide significance, but several intriguing findings emerged. Notably, we replicated associations of chromosome 9p21 with major CVD. Additional studies are needed to validate these results. Finding genetic variants associated with CVD may point to novel disease pathways and identify potential targeted preventive therapies

The Long-Baseline Neutrino Experiment: Exploring Fundamental Symmetries of the Universe

The preponderance of matter over antimatter in the early Universe, the dynamics of the supernova bursts that produced the heavy elements necessary for life and whether protons eventually decay --- these mysteries at the forefront of particle physics and astrophysics are key to understanding the early evolution of our Universe, its current state and its eventual fate. The Long-Baseline Neutrino Experiment (LBNE) represents an extensively developed plan for a world-class experiment dedicated to addressing these questions. LBNE is conceived around three central components: (1) a new, high-intensity neutrino source generated from a megawatt-class proton accelerator at Fermi National Accelerator Laboratory, (2) a near neutrino detector just downstream of the source, and (3) a massive liquid argon time-projection chamber deployed as a far detector deep underground at the Sanford Underground Research Facility. This facility, located at the site of the former Homestake Mine in Lead, South Dakota, is approximately 1,300 km from the neutrino source at Fermilab -- a distance (baseline) that delivers optimal sensitivity to neutrino charge-parity symmetry violation and mass ordering effects. This ambitious yet cost-effective design incorporates scalability and flexibility and can accommodate a variety of upgrades and contributions. With its exceptional combination of experimental configuration, technical capabilities, and potential for transformative discoveries, LBNE promises to be a vital facility for the field of particle physics worldwide, providing physicists from around the globe with opportunities to collaborate in a twenty to thirty year program of exciting science. In this document we provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess.Comment: Major update of previous version. This is the reference document for LBNE science program and current status. Chapters 1, 3, and 9 provide a comprehensive overview of LBNE's scientific objectives, its place in the landscape of neutrino physics worldwide, the technologies it will incorporate and the capabilities it will possess. 288 pages, 116 figure

The Framingham Heart Study 100K SNP genome-wide association study resource: overview of 17 phenotype working group reports

Background: The Framingham Heart Study (FHS), founded in 1948 to examine the epidemiology of cardiovascular disease, is among the most comprehensively characterized multi-generational studies in the world. Many collected phenotypes have substantial genetic contributors; yet most genetic determinants remain to be identified. Using single nucleotide polymorphisms (SNPs) from a 100K genome-wide scan, we examine the associations of common polymorphisms with phenotypic variation in this community-based cohort and provide a full-disclosure, web-based resource of results for future replication studies. Methods: Adult participants (n = 1345) of the largest 310 pedigrees in the FHS, many biologically related, were genotyped with the 100K Affymetrix GeneChip. These genotypes were used to assess their contribution to 987 phenotypes collected in FHS over 56 years of follow up, including: cardiovascular risk factors and biomarkers; subclinical and clinical cardiovascular disease; cancer and longevity traits; and traits in pulmonary, sleep, neurology, renal, and bone domains. We conducted genome-wide variance components linkage and population-based and family-based association tests. Results: The participants were white of European descent and from the FHS Original and Offspring Cohorts (examination 1 Offspring mean age 32 ± 9 years, 54% women). This overview summarizes the methods, selected findings and limitations of the results presented in the accompanying series of 17 manuscripts. The presented association results are based on 70,897 autosomal SNPs meeting the following criteria: minor allele frequency ≥ 10%, genotype call rate ≥ 80%, Hardy-Weinberg equilibrium p-value ≥ 0.001, and satisfying Mendelian consistency. Linkage analyses are based on 11,200 SNPs and short-tandem repeats. Results of phenotype-genotype linkages and associations for all autosomal SNPs are posted on the NCBI dbGaP website at http:// www.ncbi.nlm.nih.gov/projects/gap/cgi-bin/study.cgi?id=phs000007. Conclusion: We have created a full-disclosure resource of results, posted on the dbGaP website, from a genome-wide association study in the FHS. Because we used three analytical approaches to examine the association and linkage of 987 phenotypes with thousands of SNPs, our results must be considered hypothesis-generating and need to be replicated. Results from the FHS 100K project with NCBI web posting provides a resource for investigators to identify high priority findings for replication.Molecular and Cellular Biolog

Genome-wide analysis of BMI in adolescents and young adults reveals additional insight into the effects of genetic loci over the life course

Genetic loci for body mass index (BMI) in adolescence and young adulthood, a period of high risk for weight gain, are understudied, yet may yield important insight into the etiology of obesity and early intervention. To identify novel genetic loci and examine the influence of known loci on BMI during this critical time period in late adolescence and early adulthood, we performed a two-stage meta-analysis using 14 genome-wide association studies in populations of European ancestry with data on BMI between ages 16 and 25 in up to 29 880 individuals. We identified seven independent loci (P < 5.0 × 10−8) near FTO (P = 3.72 × 10−23), TMEM18 (P = 3.24 × 10−17), MC4R (P = 4.41 × 10−17), TNNI3K (P = 4.32 × 10−11), SEC16B (P = 6.24 × 10−9), GNPDA2 (P = 1.11 × 10−8) and POMC (P = 4.94 × 10−8) as well as a potential secondary signal at the POMC locus (rs2118404, P = 2.4 × 10−5 after conditioning on the established single-nucleotide polymorphism at this locus) in adolescents and young adults. To evaluate the impact of the established genetic loci on BMI at these young ages, we examined differences between the effect sizes of 32 published BMI loci in European adult populations (aged 18-90) and those observed in our adolescent and young adult meta-analysis. Four loci (near PRKD1, TNNI3K, SEC16B and CADM2) had larger effects and one locus (near SH2B1) had a smaller effect on BMI during adolescence and young adulthood compared with older adults (P < 0.05). These results suggest that genetic loci for BMI can vary in their effects across the life course, underlying the importance of evaluating BMI at different age

GENE-Counter: A Computational Pipeline for the Analysis of RNA-Seq Data for Gene Expression Differences

GENE-counter is a complete Perl-based computational pipeline for analyzing RNA-Sequencing (RNA-Seq) data for differential gene expression. In addition to its use in studying transcriptomes of eukaryotic model organisms, GENE-counter is applicable for prokaryotes and non-model organisms without an available genome reference sequence. For alignments, GENE-counter is configured for CASHX, Bowtie, and BWA, but an end user can use any Sequence Alignment/Map (SAM)-compliant program of preference. To analyze data for differential gene expression, GENE-counter can be run with any one of three statistics packages that are based on variations of the negative binomial distribution. The default method is a new and simple statistical test we developed based on an over-parameterized version of the negative binomial distribution. GENE-counter also includes three different methods for assessing differentially expressed features for enriched gene ontology (GO) terms. Results are transparent and data are systematically stored in a MySQL relational database to facilitate additional analyses as well as quality assessment. We used next generation sequencing to generate a small-scale RNA-Seq dataset derived from the heavily studied defense response of Arabidopsis thaliana and used GENE-counter to process the data. Collectively, the support from analysis of microarrays as well as the observed and substantial overlap in results from each of the three statistics packages demonstrates that GENE-counter is well suited for handling the unique characteristics of small sample sizes and high variability in gene counts