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1.  Identification of Genetic Association of Multiple Rare Variants Using Collapsing Methods 
Genetic Epidemiology  2011;35(Suppl 1):S101-S106.
Next-generation sequencing technology allows investigation of both common and rare variants in humans. Exomes are sequenced on the population level or in families to further study the genetics of human diseases. Genetic Analysis Workshop 17 (GAW17) provided exomic data from the 1000 Genomes Project and simulated phenotypes. These data enabled evaluations of existing and newly developed statistical methods for rare variant sequence analysis for which standard statistical methods fail because of the rareness of the alleles. Various alternative approaches have been proposed that overcome the rareness problem by combining multiple rare variants within a gene. These approaches are termed collapsing methods, and our GAW17 group focused on studying the performance of existing and novel collapsing methods using rare variants. All tested methods performed similarly, as measured by type I error and power. Inflated type I error fractions were consistently observed and might be caused by gametic phase disequilibrium between causal and noncausal rare variants in this relatively small sample as well as by population stratification. Incorporating prior knowledge, such as appropriate covariates and information on functionality of SNPs, increased the power of detecting associated genes. Overall, collapsing rare variants can increase the power of identifying disease-associated genes. However, studying genetic associations of rare variants remains a challenging task that requires further development and improvement in data collection, management, analysis, and computation.
doi:10.1002/gepi.20658
PMCID: PMC3289287  PMID: 22128049
1000 Genomes Project; association; collapsing methods; next-generation sequencing
2.  Two-stage study designs combining genome-wide association studies, tag single-nucleotide polymorphisms, and exome sequencing: accuracy of genetic effect estimates 
BMC Proceedings  2011;5(Suppl 9):S64.
Genome-wide association studies (GWAS) test for disease-trait associations and estimate effect sizes at tag single-nucleotide polymorphisms (SNPs), which imperfectly capture variation at causal SNPs. Sequencing studies can examine potential causal SNPs directly; however, sequencing the whole genome or exome can be prohibitively expensive. Costs can be limited by using a GWAS to detect the associated region(s) at tag SNPs followed by targeted sequencing to identify and estimate the effect size of the causal variant. Genetic effect estimates obtained from association studies can be inflated because of a form of selection bias known as the winner’s curse. Conversely, estimates at tag SNPs can be attenuated compared to the causal SNP because of incomplete linkage disequilibrium. These two effects oppose each other. Analysis of rare SNPs further complicates our understanding of the winner’s curse because rare SNPs are difficult to tag and analysis can involve collapsing over multiple rare variants. In two-stage analysis of Genetic Analysis Workshop 17 simulated data sets, we find that selection at the tag SNP produces upward bias in the estimate of effect at the causal SNP, even when the tag and causal SNPs are not well correlated. The bias similarly carries through to effect estimates for rare variant summary measures. Replication studies designed with sample sizes computed using biased estimates will be under-powered to detect a disease-causing variant. Accounting for bias in the original study is critical to avoid discarding disease-associated SNPs at follow up.
doi:10.1186/1753-6561-5-S9-S64
PMCID: PMC3287903  PMID: 22373407
3.  The Role and Challenges of Exome Sequencing in Studies of Human Diseases 
Frontiers in Genetics  2013;4:160.
Recent advances in next-generation sequencing technologies have transformed the genetics study of human diseases; this is an era of unprecedented productivity. Exome sequencing, the targeted sequencing of the protein-coding portion of the human genome, has been shown to be a powerful and cost-effective method for detection of disease variants underlying Mendelian disorders. Increasing effort has been made in the interest of the identification of rare variants associated with complex traits in sequencing studies. Here we provided an overview of the application fields for exome sequencing in human diseases. We describe a general framework of computation and bioinformatics for handling sequencing data. We then demonstrate data quality and agreement between exome sequencing and exome microarray (chip) genotypes using data collected on the same set of subjects in a genetic study of panic disorder. Our results show that, in sequencing data, the data quality was generally higher for variants within the exonic target regions, compared to that outside the target regions, due to the target enrichment. We also compared genotype concordance for variant calls obtained by exome sequencing vs. exome genotyping microarrays. The overall consistency rate was >99.83% and the heterozygous consistency rate was >97.55%. The two platforms share a large amount of agreement over low frequency variants in the exonic regions, while exome sequencing provides much more information on variants not included on exome genotyping microarrays. The results demonstrate that exome sequencing data are of high quality and can be used to investigate the role of rare coding variants in human diseases.
doi:10.3389/fgene.2013.00160
PMCID: PMC3752524  PMID: 24032039
exome sequencing; exome arrays; Mendelian diseases; complex traits; whole-genome sequencing
4.  Gene-based multiple trait analysis for exome sequencing data 
BMC Proceedings  2011;5(Suppl 9):S75.
The common genetic variants identified through genome-wide association studies explain only a small proportion of the genetic risk for complex diseases. The advancement of next-generation sequencing technologies has enabled the detection of rare variants that are expected to contribute significantly to the missing heritability. Some genetic association studies provide multiple correlated traits for analysis. Multiple trait analysis has the potential to improve the power to detect pleiotropic genetic variants that influence multiple traits. We propose a gene-level association test for multiple traits that accounts for correlation among the traits. Gene- or region-level testing for association involves both common and rare variants. Statistical tests for common variants may have limited power for individual rare variants because of their low frequency and multiple testing issues. To address these concerns, we use the weighted-sum pooling method to test the joint association of multiple rare and common variants within a gene. The proposed method is applied to the Genetic Association Workshop 17 (GAW17) simulated mini-exome data to analyze multiple traits. Because of the nature of the GAW17 simulation model, increased power was not observed for multiple-trait analysis compared to single-trait analysis. However, multiple-trait analysis did not result in a substantial loss of power because of the testing of multiple traits. We conclude that this method would be useful for identifying pleiotropic genes.
doi:10.1186/1753-6561-5-S9-S75
PMCID: PMC3287915  PMID: 22373189
5.  Identification of Grouped Rare and Common Variants via Penalized Logistic Regression 
Genetic Epidemiology  2013;37(6):592-602.
In spite of the success of genome-wide association studies in finding many common variants associated with disease, these variants seem to explain only a small proportion of the estimated heritability. Data collection has turned toward exome and whole genome sequencing, but it is well known that single marker methods frequently used for common variants have low power to detect rare variants associated with disease, even with very large sample sizes. In response, a variety of methods have been developed that attempt to cluster rare variants so that they may gather strength from one another under the premise that there may be multiple causal variants within a gene. Most of these methods group variants by gene or proximity, and test one gene or marker window at a time. We propose a penalized regression method (PeRC) that analyzes all genes at once, allowing grouping of all (rare and common) variants within a gene, along with subgrouping of the rare variants, thus borrowing strength from both rare and common variants within the same gene. The method can incorporate either a burden-based weighting of the rare variants or one in which the weights are data driven. In simulations, our method performs favorably when compared to many previously proposed approaches, including its predecessor, the sparse group lasso [Friedman et al., 2010].
doi:10.1002/gepi.21746
PMCID: PMC3842118  PMID: 23836590
penalized likelihood; lasso; elastic net; association analysis; rare variants
6.  Characterisation and Validation of Insertions and Deletions in 173 Patient Exomes 
PLoS ONE  2012;7(12):e51292.
Recent advances in genomics technologies have spurred unprecedented efforts in genome and exome re-sequencing aiming to unravel the genetic component of rare and complex disorders. While in rare disorders this allowed the identification of novel causal genes, the missing heritability paradox in complex diseases remains so far elusive. Despite rapid advances of next-generation sequencing, both the technology and the analysis of the data it produces are in its infancy. At present there is abundant knowledge pertaining to the role of rare single nucleotide variants (SNVs) in rare disorders and of common SNVs in common disorders. Although the 1,000 genome project has clearly highlighted the prevalence of rare variants and more complex variants (e.g. insertions, deletions), their role in disease is as yet far from elucidated.
We set out to analyse the properties of sequence variants identified in a comprehensive collection of exome re-sequencing studies performed on samples from patients affected by a broad range of complex and rare diseases (N = 173). Given the known potential for Loss of Function (LoF) variants to be false positive, we performed an extensive validation of the common, rare and private LoF variants identified, which indicated that most of the private and rare variants identified were indeed true, while common novel variants had a significantly higher false positive rate. Our results indicated a strong enrichment of very low-frequency insertion/deletion variants, so far under-investigated, which might be difficult to capture with low coverage and imputation approaches and for which most of study designs would be under-powered. These insertions and deletions might play a significant role in disease genetics, contributing specifically to the underlining rare and private variation predicted to be discovered through next generation sequencing.
doi:10.1371/journal.pone.0051292
PMCID: PMC3522676  PMID: 23251486
7.  A Hybrid Likelihood Model for Sequence-Based Disease Association Studies 
PLoS Genetics  2013;9(1):e1003224.
In the past few years, case-control studies of common diseases have shifted their focus from single genes to whole exomes. New sequencing technologies now routinely detect hundreds of thousands of sequence variants in a single study, many of which are rare or even novel. The limitation of classical single-marker association analysis for rare variants has been a challenge in such studies. A new generation of statistical methods for case-control association studies has been developed to meet this challenge. A common approach to association analysis of rare variants is the burden-style collapsing methods to combine rare variant data within individuals across or within genes. Here, we propose a new hybrid likelihood model that combines a burden test with a test of the position distribution of variants. In extensive simulations and on empirical data from the Dallas Heart Study, the new model demonstrates consistently good power, in particular when applied to a gene set (e.g., multiple candidate genes with shared biological function or pathway), when rare variants cluster in key functional regions of a gene, and when protective variants are present. When applied to data from an ongoing sequencing study of bipolar disorder (191 cases, 107 controls), the model identifies seven gene sets with nominal p-values0.05, of which one MAPK signaling pathway (KEGG) reaches trend-level significance after correcting for multiple testing.
Author Summary
Inexpensive, high-throughput sequencing has transformed the field of case-control association studies. For the first time, it may be possible to identify the genetic underpinnings of complex diseases, by sequencing the DNA of hundreds (even thousands) of cases and controls and comparing patterns of DNA sequence variation. However, complex diseases are likely to be caused by many variants, some of which are very rare. Taken one at a time, the association between variant and disease phenotype may not be detectable by current statistical methods. One strategy is to identify regions where important variants occur by “collapsing” variants into groups. Here, we present a new collapsing approach, capable of detecting subtle genetic differences between cases and controls. We show, in extensive simulations and using a benchmark set of genes involved in human triglyceride levels, that the approach is potentially more powerful than existing methods. We apply the new method to an ongoing sequencing study of bipolar cases and controls and identify a set of genes found in neuronal synapses, which may be implicated in bipolar disorder.
doi:10.1371/journal.pgen.1003224
PMCID: PMC3554549  PMID: 23358228
8.  A Novel Adaptive Method for the Analysis of Next-Generation Sequencing Data to Detect Complex Trait Associations with Rare Variants Due to Gene Main Effects and Interactions 
PLoS Genetics  2010;6(10):e1001156.
There is solid evidence that rare variants contribute to complex disease etiology. Next-generation sequencing technologies make it possible to uncover rare variants within candidate genes, exomes, and genomes. Working in a novel framework, the kernel-based adaptive cluster (KBAC) was developed to perform powerful gene/locus based rare variant association testing. The KBAC combines variant classification and association testing in a coherent framework. Covariates can also be incorporated in the analysis to control for potential confounders including age, sex, and population substructure. To evaluate the power of KBAC: 1) variant data was simulated using rigorous population genetic models for both Europeans and Africans, with parameters estimated from sequence data, and 2) phenotypes were generated using models motivated by complex diseases including breast cancer and Hirschsprung's disease. It is demonstrated that the KBAC has superior power compared to other rare variant analysis methods, such as the combined multivariate and collapsing and weight sum statistic. In the presence of variant misclassification and gene interaction, association testing using KBAC is particularly advantageous. The KBAC method was also applied to test for associations, using sequence data from the Dallas Heart Study, between energy metabolism traits and rare variants in ANGPTL 3,4,5 and 6 genes. A number of novel associations were identified, including the associations of high density lipoprotein and very low density lipoprotein with ANGPTL4. The KBAC method is implemented in a user-friendly R package.
Author Summary
It has been demonstrated that both rare and common variants are involved in complex disease etiology. Until recently it was only possible to perform large scale analysis of common variants. With the development of next-generation sequencing technologies, detection and mapping of rare variants have been made possible. However, methods used to analyze common variants are not powerful for the analysis of rare variants. To address the problems of rare variant analysis working in a novel framework, the kernel-based adaptive cluster (KBAC) method was developed to perform gene/locus based analysis. The KBAC combines variant classification and association testing in a coherent framework. Through simulations motivated by population genetic and disease data, it is demonstrated that the KBAC has superior power to other rare variant analysis methods, especially in the presence of variant misclassification and gene interaction. Using data from the Dallas Heart Study, the KBAC method was applied to test for associations between energy metabolism traits and rare variants in ANGPTL 3,4,5 and 6 genes. A number of novel associations were identified. The KBAC method is implemented in a user-friendly R package.
doi:10.1371/journal.pgen.1001156
PMCID: PMC2954824  PMID: 20976247
9.  Combining effects from rare and common genetic variants in an exome-wide association study of sequence data 
BMC Proceedings  2011;5(Suppl 9):S44.
Recent breakthroughs in next-generation sequencing technologies allow cost-effective methods for measuring a growing list of cellular properties, including DNA sequence and structural variation. Next-generation sequencing has the potential to revolutionize complex trait genetics by directly measuring common and rare genetic variants within a genome-wide context. Because for a given gene both rare and common causal variants can coexist and have independent effects on a trait, strategies that model the effects of both common and rare variants could enhance the power of identifying disease-associated genes. To date, little work has been done on integrating signals from common and rare variants into powerful statistics for finding disease genes in genome-wide association studies. In this analysis of the Genetic Analysis Workshop 17 data, we evaluate various strategies for association of rare, common, or a combination of both rare and common variants on quantitative phenotypes in unrelated individuals. We show that the analysis of common variants only using classical approaches can achieve higher power to detect causal genes than recently proposed rare variant methods and that strategies that combine association signals derived independently in rare and common variants can slightly increase the power compared to strategies that focus on the effect of either the rare variants or the common variants.
doi:10.1186/1753-6561-5-S9-S44
PMCID: PMC3287881  PMID: 22373328
10.  Quality Control Issues and the Identification of Rare Functional Variants with Next-Generation Sequencing Data 
Genetic Epidemiology  2011;35(Suppl 1):S22-S28.
Next-generation sequencing of large numbers of individuals presents challenges in data preparation, quality control, and statistical analysis because of the rarity of the variants. The Genetic Analysis Workshop 17 (GAW17) data provide an opportunity to survey existing methods and compare these methods with novel ones. Specifically, the GAW17 Group 2 contributors investigate existing and newly proposed methods and study design strategies to identify rare variants, predict functional variants, and/or examine quality control. We introduce the eight Group 2 papers, summarize their approaches, and discuss their strengths and weaknesses. For these investigations, some groups used only the genotype data, whereas others also used the simulated phenotype data. Although the eight Group 2 contributions covered a wide variety of topics under the general idea of identifying rare variants, they can be grouped into three broad categories according to their common research interests: functionality of variants and quality control issues, family-based analyses, and association analyses of unrelated individuals. The aims of the first subgroup were quite different. These were population structure analyses that used rare variants to predict functionality and examine the accuracy of genotype calls. The aims of the family-based analyses were to select which families should be sequenced and to identify high-risk pedigrees; the aim of the association analyses was to identify variants or genes with regression-based methods. However, power to detect associations was low in all three association studies. Thus this work shows opportunities for incorporating rare variants into the genetic and statistical analyses of common diseases.
doi:10.1002/gepi.20645
PMCID: PMC3268158  PMID: 22128054
1000 Genomes Project; association; collection of rare variants; family data; next-generation sequencing; regression; quality control
11.  Bayesian Detection of Causal Rare Variants under Posterior Consistency 
PLoS ONE  2013;8(7):e69633.
Identification of causal rare variants that are associated with complex traits poses a central challenge on genome-wide association studies. However, most current research focuses only on testing the global association whether the rare variants in a given genomic region are collectively associated with the trait. Although some recent work, e.g., the Bayesian risk index method, have tried to address this problem, it is unclear whether the causal rare variants can be consistently identified by them in the small--large- situation. We develop a new Bayesian method, the so-called Bayesian Rare Variant Detector (BRVD), to tackle this problem. The new method simultaneously addresses two issues: (i) (Global association test) Are there any of the variants associated with the disease, and (ii) (Causal variant detection) Which variants, if any, are driving the association. The BRVD ensures the causal rare variants to be consistently identified in the small--large- situation by imposing some appropriate prior distributions on the model and model specific parameters. The numerical results indicate that the BRVD is more powerful for testing the global association than the existing methods, such as the combined multivariate and collapsing test, weighted sum statistic test, RARECOVER, sequence kernel association test, and Bayesian risk index, and also more powerful for identification of causal rare variants than the Bayesian risk index method. The BRVD has also been successfully applied to the Early-Onset Myocardial Infarction (EOMI) Exome Sequence Data. It identified a few causal rare variants that have been verified in the literature.
doi:10.1371/journal.pone.0069633
PMCID: PMC3724943  PMID: 23922764
12.  GUESS-ing Polygenic Associations with Multiple Phenotypes Using a GPU-Based Evolutionary Stochastic Search Algorithm 
PLoS Genetics  2013;9(8):e1003657.
Genome-wide association studies (GWAS) yielded significant advances in defining the genetic architecture of complex traits and disease. Still, a major hurdle of GWAS is narrowing down multiple genetic associations to a few causal variants for functional studies. This becomes critical in multi-phenotype GWAS where detection and interpretability of complex SNP(s)-trait(s) associations are complicated by complex Linkage Disequilibrium patterns between SNPs and correlation between traits. Here we propose a computationally efficient algorithm (GUESS) to explore complex genetic-association models and maximize genetic variant detection. We integrated our algorithm with a new Bayesian strategy for multi-phenotype analysis to identify the specific contribution of each SNP to different trait combinations and study genetic regulation of lipid metabolism in the Gutenberg Health Study (GHS). Despite the relatively small size of GHS (n = 3,175), when compared with the largest published meta-GWAS (n>100,000), GUESS recovered most of the major associations and was better at refining multi-trait associations than alternative methods. Amongst the new findings provided by GUESS, we revealed a strong association of SORT1 with TG-APOB and LIPC with TG-HDL phenotypic groups, which were overlooked in the larger meta-GWAS and not revealed by competing approaches, associations that we replicated in two independent cohorts. Moreover, we demonstrated the increased power of GUESS over alternative multi-phenotype approaches, both Bayesian and non-Bayesian, in a simulation study that mimics real-case scenarios. We showed that our parallel implementation based on Graphics Processing Units outperforms alternative multi-phenotype methods. Beyond multivariate modelling of multi-phenotypes, our Bayesian model employs a flexible hierarchical prior structure for genetic effects that adapts to any correlation structure of the predictors and increases the power to identify associated variants. This provides a powerful tool for the analysis of diverse genomic features, for instance including gene expression and exome sequencing data, where complex dependencies are present in the predictor space.
Author Summary
Nowadays, the availability of cheaper and accurate assays to quantify multiple (endo)phenotypes in large population cohorts allows multi-trait studies. However, these studies are limited by the lack of flexible models integrated with efficient computational tools for genome-wide multi SNPs-traits analyses. To overcome this problem, we propose a novel Bayesian analysis strategy and a new algorithmic implementation which exploits parallel processing architecture for fully multivariate modeling of groups of correlated phenotypes at the genome-wide scale. In addition to increased power of our algorithm over alternative Bayesian and well-established non-Bayesian multi-phenotype methods, we provide an application to a real case study of several blood lipid traits, and show how our method recovered most of the major associations and is better at refining multi-trait polygenic associations than alternative methods. We reveal and replicate in independent cohorts new associations with two phenotypic groups that were not detected by competing multivariate approaches and not noticed by a large meta-GWAS. We also discuss the applicability of the proposed method to large meta-analyses involving hundreds of thousands of individuals and to diverse genomic datasets where complex dependencies in the predictor space are present.
doi:10.1371/journal.pgen.1003657
PMCID: PMC3738451  PMID: 23950726
13.  Identification of multiple rare variants associated with a disease 
BMC Proceedings  2011;5(Suppl 9):S103.
Identifying rare variants that are responsible for complex disease has been promoted by advances in sequencing technologies. However, statistical methods that can handle the vast amount of data generated and that can interpret the complicated relationship between disease and these variants have lagged. We apply a zero-inflated Poisson regression model to take into account the excess of zeros caused by the extremely low frequency of the 24,487 exonic variants in the Genetic Analysis Workshop 17 data. We grouped the 697 subjects in the data set as Europeans, Asians, and Africans based on principal components analysis and found the total number of rare variants per gene for each individual. We then analyzed these collapsed variants based on the assumption that rare variants are enriched in a group of people affected by a disease compared to a group of unaffected people. We also tested the hypothesis with quantitative traits Q1, Q2, and Q4. Analyses performed on the combined 697 individuals and on each ethnic group yielded different results. For the combined population analysis, we found that UGT1A1, which was not part of the simulation model, was associated with disease liability and that FLT1, which was a causal locus in the simulation model, was associated with Q1. Of the causal loci in the simulation models, FLT1 and KDR were associated with Q1 and VNN1 was correlated with Q2. No significant genes were associated with Q4. These results show the feasibility and capability of our new statistical model to detect multiple rare variants influencing disease risk.
doi:10.1186/1753-6561-5-S9-S103
PMCID: PMC3287826  PMID: 22373445
14.  Genetic Architecture of Vitamin B12 and Folate Levels Uncovered Applying Deeply Sequenced Large Datasets 
PLoS Genetics  2013;9(6):e1003530.
Genome-wide association studies have mainly relied on common HapMap sequence variations. Recently, sequencing approaches have allowed analysis of low frequency and rare variants in conjunction with common variants, thereby improving the search for functional variants and thus the understanding of the underlying biology of human traits and diseases. Here, we used a large Icelandic whole genome sequence dataset combined with Danish exome sequence data to gain insight into the genetic architecture of serum levels of vitamin B12 (B12) and folate. Up to 22.9 million sequence variants were analyzed in combined samples of 45,576 and 37,341 individuals with serum B12 and folate measurements, respectively. We found six novel loci associating with serum B12 (CD320, TCN2, ABCD4, MMAA, MMACHC) or folate levels (FOLR3) and confirmed seven loci for these traits (TCN1, FUT6, FUT2, CUBN, CLYBL, MUT, MTHFR). Conditional analyses established that four loci contain additional independent signals. Interestingly, 13 of the 18 identified variants were coding and 11 of the 13 target genes have known functions related to B12 and folate pathways. Contrary to epidemiological studies we did not find consistent association of the variants with cardiovascular diseases, cancers or Alzheimer's disease although some variants demonstrated pleiotropic effects. Although to some degree impeded by low statistical power for some of these conditions, these data suggest that sequence variants that contribute to the population diversity in serum B12 or folate levels do not modify the risk of developing these conditions. Yet, the study demonstrates the value of combining whole genome and exome sequencing approaches to ascertain the genetic and molecular architectures underlying quantitative trait associations.
Author Summary
Genome-wide association studies have in recent years revealed a wealth of common variants associated with common diseases and phenotypes. We took advantage of the advances in sequencing technologies to study the association of low frequency and rare variants in conjunction with common variants with serum levels of vitamin B12 (B12) and folate in Icelanders and Danes. We found 18 independent signals in 13 loci associated with serum B12 or folate levels. Interestingly, 13 of the 18 identified variants are coding and 11 of the 13 target genes have known functions related to B12 and folate pathways. These data indicate that the target genes at all of the loci have been identified. Epidemiological studies have shown a relationship between serum B12 and folate levels and the risk of cardiovascular diseases, cancers, and Alzheimer's disease. We investigated association between the identified variants and these diseases but did not find consistent association.
doi:10.1371/journal.pgen.1003530
PMCID: PMC3674994  PMID: 23754956
15.  Identifying Mendelian disease genes with the Variant Effect Scoring Tool 
BMC Genomics  2013;14(Suppl 3):S3.
Background
Whole exome sequencing studies identify hundreds to thousands of rare protein coding variants of ambiguous significance for human health. Computational tools are needed to accelerate the identification of specific variants and genes that contribute to human disease.
Results
We have developed the Variant Effect Scoring Tool (VEST), a supervised machine learning-based classifier, to prioritize rare missense variants with likely involvement in human disease. The VEST classifier training set comprised ~ 45,000 disease mutations from the latest Human Gene Mutation Database release and another ~45,000 high frequency (allele frequency >1%) putatively neutral missense variants from the Exome Sequencing Project. VEST outperforms some of the most popular methods for prioritizing missense variants in carefully designed holdout benchmarking experiments (VEST ROC AUC = 0.91, PolyPhen2 ROC AUC = 0.86, SIFT4.0 ROC AUC = 0.84). VEST estimates variant score p-values against a null distribution of VEST scores for neutral variants not included in the VEST training set. These p-values can be aggregated at the gene level across multiple disease exomes to rank genes for probable disease involvement. We tested the ability of an aggregate VEST gene score to identify candidate Mendelian disease genes, based on whole-exome sequencing of a small number of disease cases. We used whole-exome data for two Mendelian disorders for which the causal gene is known. Considering only genes that contained variants in all cases, the VEST gene score ranked dihydroorotate dehydrogenase (DHODH) number 2 of 2253 genes in four cases of Miller syndrome, and myosin-3 (MYH3) number 2 of 2313 genes in three cases of Freeman Sheldon syndrome.
Conclusions
Our results demonstrate the potential power gain of aggregating bioinformatics variant scores into gene-level scores and the general utility of bioinformatics in assisting the search for disease genes in large-scale exome sequencing studies. VEST is available as a stand-alone software package at http://wiki.chasmsoftware.org and is hosted by the CRAVAT web server at http://www.cravat.us
doi:10.1186/1471-2164-14-S3-S3
PMCID: PMC3665549  PMID: 23819870
16.  Best Practices and Joint Calling of the HumanExome BeadChip: The CHARGE Consortium 
PLoS ONE  2013;8(7):e68095.
Genotyping arrays are a cost effective approach when typing previously-identified genetic polymorphisms in large numbers of samples. One limitation of genotyping arrays with rare variants (e.g., minor allele frequency [MAF] <0.01) is the difficulty that automated clustering algorithms have to accurately detect and assign genotype calls. Combining intensity data from large numbers of samples may increase the ability to accurately call the genotypes of rare variants. Approximately 62,000 ethnically diverse samples from eleven Cohorts for Heart and Aging Research in Genomic Epidemiology (CHARGE) Consortium cohorts were genotyped with the Illumina HumanExome BeadChip across seven genotyping centers. The raw data files for the samples were assembled into a single project for joint calling. To assess the quality of the joint calling, concordance of genotypes in a subset of individuals having both exome chip and exome sequence data was analyzed. After exclusion of low performing SNPs on the exome chip and non-overlap of SNPs derived from sequence data, genotypes of 185,119 variants (11,356 were monomorphic) were compared in 530 individuals that had whole exome sequence data. A total of 98,113,070 pairs of genotypes were tested and 99.77% were concordant, 0.14% had missing data, and 0.09% were discordant. We report that joint calling allows the ability to accurately genotype rare variation using array technology when large sample sizes are available and best practices are followed. The cluster file from this experiment is available at www.chargeconsortium.com/main/exomechip.
doi:10.1371/journal.pone.0068095
PMCID: PMC3709915  PMID: 23874508
17.  Genetic complexity in hypertrophic cardiomyopathy revealed by high-throughput sequencing 
Journal of Medical Genetics  2013;50(4):228-239.
Background
Clinical interpretation of the large number of rare variants identified by high throughput sequencing (HTS) technologies is challenging. The aim of this study was to explore the clinical implications of a HTS strategy for patients with hypertrophic cardiomyopathy (HCM) using a targeted HTS methodology and workflow developed for patients with a range of inherited cardiovascular diseases. By comparing the sequencing results with published findings and with sequence data from a large-scale exome sequencing screen of UK individuals, we sought to quantify the strength of the evidence supporting causality for detected candidate variants.
Methods and results
223 unrelated patients with HCM (46±15 years at diagnosis, 74% males) were studied. In order to analyse coding, intronic and regulatory regions of 41 cardiovascular genes, we used solution-based sequence capture followed by massive parallel resequencing on Illumina GAIIx. Average read-depth in the 2.1 Mb target region was 120. Rare (frequency<0.5%) non-synonymous, loss-of-function and splice-site variants were defined as candidates. Excluding titin, we identified 152 distinct candidate variants in sarcomeric or associated genes (89 novel) in 143 patients (64%). Four sarcomeric genes (MYH7, MYBPC3, TNNI3, TNNT2) showed an excess of rare single non-synonymous single-nucleotide polymorphisms (nsSNPs) in cases compared to controls. The estimated probability that a nsSNP in these genes is pathogenic varied between 57% and near certainty depending on the location. We detected an additional 94 candidate variants (73 novel) in desmosomal, and ion-channel genes in 96 patients (43%).
Conclusions
This study provides the first large-scale quantitative analysis of the prevalence of sarcomere protein gene variants in patients with HCM using HTS technology. Inclusion of other genes implicated in inherited cardiac disease identifies a large number of non-synonymous rare variants of unknown clinical significance.
doi:10.1136/jmedgenet-2012-101270
PMCID: PMC3607113  PMID: 23396983
Hypertrophic Cardiomyopathy; Genetics; High-throughput sequencing
18.  Exploring the potential benefits of stratified false discovery rates for region-based testing of association with rare genetic variation 
When analyzing the data that arises from exome or whole-genome sequencing studies, window-based tests, (i.e., tests that jointly analyze all genetic data in a small genomic region), are very popular. However, power is known to be quite low for finding associations with phenotypes using these tests, and therefore a variety of analytic strategies may be employed to potentially improve power. Using sequencing data of all of chromosome 3 from an interim release of data on 2432 individuals from the UK10K project, we simulated phenotypes associated with rare genetic variation, and used the results to explore the window-based test power. We asked two specific questions: firstly, whether there could be substantial benefits associated with incorporating information from external annotation on the genetic variants, and secondly whether the false discovery rate (FDRs) would be a useful metric for assessing significance. Although, as expected, there are benefits to using additional information (such as annotation) when it is associated with causality, we confirmed the general pattern of low sensitivity and power for window-based tests. For our chosen example, even when power is high to detect some of the associations, many of the regions containing causal variants are not detectable, despite using lax significance thresholds and optimal analytic methods. Furthermore, our estimated FDR values tended to be much smaller than the true FDRs. Long-range correlations between variants—due to linkage disequilibrium—likely explain some of this bias. A more sophisticated approach to using the annotation information may improve power, however, many causal variants of realistic effect sizes may simply be undetectable, at least with this sample size. Perhaps annotation information could assist in distinguishing windows containing causal variants from windows that are merely correlated with causal variants.
doi:10.3389/fgene.2014.00011
PMCID: PMC3905218  PMID: 24523729
rare genetic variants; SNV; false discovery rate; multiple testing; genomic annotation; whole genome sequencing; window-based tests; stratified false discovery rate
19.  A Flexible Approach for the Analysis of Rare Variants Allowing for a Mixture of Effects on Binary or Quantitative Traits 
PLoS Genetics  2013;9(8):e1003694.
Multiple rare variants either within or across genes have been hypothesised to collectively influence complex human traits. The increasing availability of high throughput sequencing technologies offers the opportunity to study the effect of rare variants on these traits. However, appropriate and computationally efficient analytical methods are required to account for collections of rare variants that display a combination of protective, deleterious and null effects on the trait. We have developed a novel method for the analysis of rare genetic variation in a gene, region or pathway that, by simply aggregating summary statistics at each variant, can: (i) test for the presence of a mixture of effects on a trait; (ii) be applied to both binary and quantitative traits in population-based and family-based data; (iii) adjust for covariates to allow for non-genetic risk factors and; (iv) incorporate imputed genetic variation. In addition, for preliminary identification of promising genes, the method can be applied to association summary statistics, available from meta-analysis of published data, for example, without the need for individual level genotype data. Through simulation, we show that our method is immune to the presence of bi-directional effects, with no apparent loss in power across a range of different mixtures, and can achieve greater power than existing approaches as long as summary statistics at each variant are robust. We apply our method to investigate association of type-1 diabetes with imputed rare variants within genes in the major histocompatibility complex using genotype data from the Wellcome Trust Case Control Consortium.
Author Summary
Rapid advances in sequencing technology mean that it is now possible to directly assay rare genetic variation. In addition, the availability of almost fully sequenced human genomes by the 1000 Genomes Project allows genotyping at rare variants that are not present on arrays commonly used in genome-wide association studies. Rare variants within a gene or region may act to collectively influence a complex trait. Methods for testing these rare variants should be able to account for a combination of those that serve to either increase, decrease or have no effect on the trait of interest. Here, we introduce a method for the analysis of a collection of rare genetic variants, within a gene or region, which assesses evidence for a mixture of effects. Our method simply aggregates summary statistics at each variant and, as such, can be applied to both population and family-based data, to binary or quantitative traits and to either directly genotyped or imputed data. In addition, it does not require individual level genotype or phenotype data, and can be adjusted for non-genetic risk factors. We illustrate our approach by examining imputed rare variants in the major histocompatibility complex for association with type-1 diabetes using genotype data from the Wellcome Trust case Control Consortium.
doi:10.1371/journal.pgen.1003694
PMCID: PMC3744430  PMID: 23966874
20.  Integrating Multiple Genomic Data to Predict Disease-Causing Nonsynonymous Single Nucleotide Variants in Exome Sequencing Studies 
PLoS Genetics  2014;10(3):e1004237.
Exome sequencing has been widely used in detecting pathogenic nonsynonymous single nucleotide variants (SNVs) for human inherited diseases. However, traditional statistical genetics methods are ineffective in analyzing exome sequencing data, due to such facts as the large number of sequenced variants, the presence of non-negligible fraction of pathogenic rare variants or de novo mutations, and the limited size of affected and normal populations. Indeed, prevalent applications of exome sequencing have been appealing for an effective computational method for identifying causative nonsynonymous SNVs from a large number of sequenced variants. Here, we propose a bioinformatics approach called SPRING (Snv PRioritization via the INtegration of Genomic data) for identifying pathogenic nonsynonymous SNVs for a given query disease. Based on six functional effect scores calculated by existing methods (SIFT, PolyPhen2, LRT, MutationTaster, GERP and PhyloP) and five association scores derived from a variety of genomic data sources (gene ontology, protein-protein interactions, protein sequences, protein domain annotations and gene pathway annotations), SPRING calculates the statistical significance that an SNV is causative for a query disease and hence provides a means of prioritizing candidate SNVs. With a series of comprehensive validation experiments, we demonstrate that SPRING is valid for diseases whose genetic bases are either partly known or completely unknown and effective for diseases with a variety of inheritance styles. In applications of our method to real exome sequencing data sets, we show the capability of SPRING in detecting causative de novo mutations for autism, epileptic encephalopathies and intellectual disability. We further provide an online service, the standalone software and genome-wide predictions of causative SNVs for 5,080 diseases at http://bioinfo.au.tsinghua.edu.cn/spring.
Author Summary
The detection of causative nonsynonymous single nucleotide variants (SNVs) is essential for the understanding of the pathogenesis of human inherited diseases. In this paper, we propose a statistical method called SPRING (Snv PRioritization via the INtegration of Genomic data) to combine six functional effect scores calculated by existing methods and five association scores derived from multiple genomic data sources to estimate the statistical significance that a nonsynonymous SNV is pathogenic for a query disease. We find that SPRING is effective in identifying disease-causing SNVs for diseases whose genetic bases are either partly known or completely unknown across a variety of inheritance styles. With real exome sequencing data, we show the qualified potential of SPRING in not only the detection of causative SNVs in simulation studies but also the identification of pathogenic de novo mutations for autism, epileptic encephalopathies and intellectual disability.
doi:10.1371/journal.pgen.1004237
PMCID: PMC3961190  PMID: 24651380
21.  Molecular Genetic Studies of Complex Phenotypes 
Translational Research  2011;159(2):64-79.
The approach to molecular genetic studies of complex phenotypes has evolved considerably during the recent years. The candidate gene approach, restricted to analysis of a few single nucleotide polymorphisms (SNPs) in a modest number of cases and controls, has been supplanted by the unbiased approach of Genome-Wide Association Studies (GWAS), wherein a large number of tagger SNPs are typed in a large number of individuals. GWAS, which are designed upon the common disease- common variant hypothesis (CD-CV), have identified a large number of SNPs and loci for complex phenotypes. However, alleles identified through GWAS are typically not causative but rather in linkage disequilibrium (LD) with the true causal variants. The common alleles, which may not capture the uncommon and rare variants, account only for a fraction of heritability of the complex traits. Hence, the focus is being shifted to rare variants – common disease (RV-CD) hypothesis, surmising that rare variants exert large effect sizes on the phenotype. In conjunctional with this conceptual shift technological advances in DNA sequencing techniques have dramatically enhanced whole genome or whole exome sequencing capacity. The sequencing approach affords identification of not only the rare but also the common variants. The approach – whether used in complementation with GWAS or as a stand-alone approach - could define the genetic architecture of the complex phenotypes. Robust phenotyping and large-scale sequencing studies are essential to extract the information content of the vast number of DNA sequence variants (DSVs) in the genome. To garner meaningful clinical information and link the genotype to a phenotype, identification and characterization of a very large number of causal fields beyond the information content of DNA sequence variants would be necessary. This review provides an update on the current progress and limitations in identifying DSVs that are associated with phenotypic effects.
doi:10.1016/j.trsl.2011.08.001
PMCID: PMC3259530  PMID: 22243791
22.  The Empirical Power of Rare Variant Association Methods: Results from Sanger Sequencing in 1,998 Individuals 
PLoS Genetics  2012;8(2):e1002496.
The role of rare genetic variation in the etiology of complex disease remains unclear. However, the development of next-generation sequencing technologies offers the experimental opportunity to address this question. Several novel statistical methodologies have been recently proposed to assess the contribution of rare variation to complex disease etiology. Nevertheless, no empirical estimates comparing their relative power are available. We therefore assessed the parameters that influence their statistical power in 1,998 individuals Sanger-sequenced at seven genes by modeling different distributions of effect, proportions of causal variants, and direction of the associations (deleterious, protective, or both) in simulated continuous trait and case/control phenotypes. Our results demonstrate that the power of recently proposed statistical methods depend strongly on the underlying hypotheses concerning the relationship of phenotypes with each of these three factors. No method demonstrates consistently acceptable power despite this large sample size, and the performance of each method depends upon the underlying assumption of the relationship between rare variants and complex traits. Sensitivity analyses are therefore recommended to compare the stability of the results arising from different methods, and promising results should be replicated using the same method in an independent sample. These findings provide guidance in the analysis and interpretation of the role of rare base-pair variation in the etiology of complex traits and diseases.
Author Summary
There is now evidence that rare variants can contribute to the etiology of complex disease. Next generation sequencing technologies have enabled their detection in large cohorts, and new statistical methods have been proposed to ascertain their association with complex diseases and traits in order to improve power over single-marker analysis. Each of these new methods assumes a particular nature of the relationship between rare variants and complex disease, yet these hypotheses have been largely unverified. Therefore we sought to compare the power of commonly used and novel statistical methods for rare variants using Sanger sequencing data from 1,998 individuals sequenced at 7 genes by simulating several phenotypes under models spanning a spectrum of the common hypotheses concerning such associations. While all methods perform reasonably well under their own model-specific hypotheses, no single method gives consistently acceptable power when these hypotheses are violated. Unlike GWAS, wherein all variants can often be tested using the same method across the entire genome, the analysis and interpretation of sequencing studies will therefore be considerably more challenging.
doi:10.1371/journal.pgen.1002496
PMCID: PMC3271058  PMID: 22319458
23.  Exome sequencing and the genetic basis of complex traits 
Nature genetics  2012;44(6):623-630.
Exome sequencing is emerging as a popular approach to study the effect of rare coding variants on complex phenotypes. The promise of exome sequencing is grounded in theoretical population genetics and in empirical successes of candidate gene sequencing studies. Many projects aimed at common diseases are underway, and their results are eagerly anticipated. In this Perspective, using exome sequencing data from 438 individuals, we discuss several aspects of exome sequencing studies that we view as particularly important. We review processing and quality control of raw sequence data, evaluate the statistical properties of exome sequencing studies, discuss rare variant burden tests to detect association to phenotypes, and demonstrate the importance of accounting for population stratification in the analysis of rare variants. We conclude that enthusiasm for exome sequencing studies of complex traits should be combined with the caution that thousands of samples may be required to reach sufficient statistical power.
doi:10.1038/ng.2303
PMCID: PMC3727622  PMID: 22641211
24.  In search of low-frequency and rare variants affecting complex traits 
Human Molecular Genetics  2013;22(R1):R16-R21.
The allelic architecture of complex traits is likely to be underpinned by a combination of multiple common frequency and rare variants. Targeted genotyping arrays and next-generation sequencing technologies at the whole-genome sequencing (WGS) and whole-exome scales (WES) are increasingly employed to access sequence variation across the full minor allele frequency (MAF) spectrum. Different study design strategies that make use of diverse technologies, imputation and sample selection approaches are an active target of development and evaluation efforts. Initial insights into the contribution of rare variants in common diseases and medically relevant quantitative traits point to low-frequency and rare alleles acting either independently or in aggregate and in several cases alongside common variants. Studies conducted in population isolates have been successful in detecting rare variant associations with complex phenotypes. Statistical methodologies that enable the joint analysis of rare variants across regions of the genome continue to evolve with current efforts focusing on incorporating information such as functional annotation, and on the meta-analysis of these burden tests. In addition, population stratification, defining genome-wide statistical significance thresholds and the design of appropriate replication experiments constitute important considerations for the powerful analysis and interpretation of rare variant association studies. Progress in addressing these emerging challenges and the accrual of sufficiently large data sets are poised to help the field of complex trait genetics enter a promising era of discovery.
doi:10.1093/hmg/ddt376
PMCID: PMC3782074  PMID: 23922232
25.  Propensity score analysis in the Genetic Analysis Workshop 17 simulated data set on independent individuals 
BMC Proceedings  2011;5(Suppl 9):S71.
Genetic Analysis Workshop 17 provided simulated phenotypes and exome sequence data for 697 independent individuals (209 case subjects and 488 control subjects). The disease liability in these data was influenced by multiple quantitative traits. We addressed the lack of statistical power in this small data set by limiting the genomic variants included in the study to those with potential disease-causing effect, thereby reducing the problem of multiple testing. After this adjustment, we could readily detect two common variants that were strongly associated with the quantitative trait Q1 (C13S523 and C13S522). However, we found no significant associations with the affected status or with any of the other quantitative traits, and the relationship between disease status and genomic variants remained obscure. To address the challenge of the multivariate phenotype, we used propensity scores to combine covariates with genetic risk factors into a single risk factor and created a new phenotype variable, the probability of being affected given the covariates. Using the propensity score as a quantitative trait in the case-control analysis, we again could identify the two common single-nucleotide polymorphisms (C13S523 and C13S522). In addition, this analysis captured the correlation between Q1 and the affected status and reduced the problem of multiple testing. Although the propensity score was useful for capturing and clarifying the genetic contributions of common variants to the disease phenotype and the mediating role of the quantitative trait Q1, the analysis did not increase power to detect rare variants.
doi:10.1186/1753-6561-5-S9-S71
PMCID: PMC3287911  PMID: 22373075

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