Introduction

Currently there is great interest in investigating the etiology of complex disease due to rare variants [1]–[6]. Until recently, indirect mapping of common variants has been the emphasis of complex trait association studies. It has been demonstrated that common variants tend to have modest phenotypic effects while rare variants are likely to have stronger phenotypic effects [7], although not strong enough to cause familial aggregation [8]. For mapping complex diseases due to common variants, instead of genotyping functional variants, tagSNPs are genotyped which act as a proxy for the underlying causal variants. For rare variant association studies, indirect mapping is not an optimal approach due to low correlations () between tagSNPs and rare variants. Instead, direct mapping should be used, where functional variants are analyzed. In order to implement direct mapping, variants must first be identified. Large scale sequencing efforts have begun including the 1000 Genome Project, which will provide a better understanding of the allelic architecture of the genome and a detailed catalog of human variants. Next-generation sequencing technologies e.g. Roche 454, ABI SOLiD, and Illumina HiSeq, have made it feasible to carry-out rare variant association studies of candidate regions, exomes and genomes.

Gene interactions are believed to be involved in a broad spectrum of complex disease etiologies [9]. Although a number of methods have been developed to detect gene interactions between common variants [10]–[13], their detection has been limited [10]. There is evidence that rare variant interaction also plays a role in disease etiology. In direct association mapping of rare variants, one or more genetic loci are commonly jointly analyzed in order to aggregate information, for example genes with similar functions or residing in the same pathway [3], [4]. Therefore it is necessary to account for potential interactions between rare variants in different loci [14] and interactions between common and rare variants [15], [16].

Ideally, when carrying out direct mapping, only causal variants should be tested for associations. When DNA samples are sequenced, both causal and non-causal variants are uncovered. Bioinformatics tools [17], [18] or filters [1] can be used to predict functionality of variants, although tools such as PolyPhen [18] or SIFT [17]can have low sensitivity and specificity [6], [19]. Empirical studies have shown that predictive errors can be as high as 47% and 37% for PolyPhen and SIFT respectively [6]; therefore, their usefulness in selecting variants to be included in association analysis is limited. Even when functionality can be correctly inferred, whether the identified variants affect the phenotype of interest is still unknown. Two types of misclassifications of variant causality can frequently arise: 1.) non-causal variants are included in the analysis: a.) sequencing incorrectly identifies monomorphic sites as variant sites (false positive SNP discovery), b.) variants are falsely predicted to be functional or c.) variants are functional but non-causal; 2.) causal variants are excluded from the analysis: a.) due to locus heterogeneity, not all loci containing causal variants are included in the analysis, b.) region not sequenced, e.g. intronic variants, c.) variants not detected by sequencing assay (false negative SNP discovery) or d.) causal variants are falsely predicted to be non-functional.

Driven by the advancement of sequencing technologies and availability of data, statistical and computational methods are needed for analyzing sequence data. It has been demonstrated that methods used to analyze common variants are low powered when applied to the analysis of rare variants [20], [21]. Methods to analyze rare variants have been proposed [20], [21]; although they have clear advantages over implementing common variant analysis approaches, more powerful and robust methods need to be developed to analyze rare variant data, especially in the presence of variant misclassification or gene interactions.

The Kernel Based Adaptive Cluster (KBAC) was developed to overcome the problems of detecting rare variant associations in the presence of misclassification and gene interaction. Under the KBAC framework, data-based adaptive variant classification and testing of association are unified. The sample risk of a multi-site genotype is modeled using a mixture distribution with two components, where one component represents the distribution of sample risk of genotype if it is non-causal and the other component represents distribution of sample risks of causal genotypes. Ideally, if distributions for causal components were known, classification could first be performed and only the causal genotypes would be used in association studies. However, when searching for genotype-phenotype associations, it is usually unknown which variants are causal. Instead of performing an unrealistic two-step procedure, variant classification and association testing are unified in the KBAC framework. Continuous adaptive weighting which is implemented in the KBAC is preferable, particularly for low frequency alleles, than classifying variants and carrying out a stratified analysis, because increasing classification and shrinking size of strata can increase both type I and II error. For the KBAC, adaptive weighting procedure is implemented using the cumulative distribution functions for the multi-site genotype counts. Distributions of multi-site genotype counts are compared between cases and controls. Those multi-site genotypes that are enriched in cases will be up-weighted. Under the null hypothesis, the assigned weights asymptotically follow a uniform distribution. While under the alternative hypothesis, disease causal multi-site genotypes tend to be more frequent in cases than in controls. Therefore they are more likely to be adaptively up-weighted. The weighted multi-site genotype frequencies are aggregated and contrasted between cases and controls. In order to evaluate whether there is an association, significance of the KBAC can be assessed using either permutation or Monte Carlo approximation (See Methods and Figure S1).

The performance of the KBAC was compared to the weighted sum statistic (WSS) [21] the combined multivariate and collapsing (CMC) method [20], and the comparison of rare variants found exclusively in cases to those found only in controls (RVE) [3] using simulated data sets. Forward time simulation [22] assuming infinite-site Wright-Fisher model was used to generate population genetic data. Demographic change and purifying selection were both incorporated in the simulation, using parameters estimated from re-sequencing datasets from studies of African Americans (AA) and European Americans (EA) [23]. In addition to forward time simulation, population genetic data was also generated according to estimated site frequency spectrums (SFS) in AA and EA from the Dallas Heart Study (DHS) re-sequencing data of the ANGPTL3, 4, 5, and 6 genes.

For the simulated population data phenotypes were generated separately and motivated by epidemiological disease studies. Two types of main effects phenotypic model are considered: 1.) constant genetic effects for each causal variant and 2.) genetic effects inversely correlated with minor allele frequencies (MAF) of causal genetic variants. In order to evaluate the impact of variant misclassification, a variety of scenarios were examined where 1.) different proportions of non-causal variants were included in the analysis and 2.) different proportions of causal variants were excluded from the analysis.

Two disease models of gene interactions were also evaluated. The example of with-in gene interaction was motivated by Hirschsprung's disease [15], [16], where an interaction between a common polymorphism in the promoter region and multiple rare non-synonymous (NS) mutations in exonic regions of the RET gene is hypothesized [15], [16]. The example of between gene interaction is based on the observation that rare variants within the CHEK2 gene increase risk of breast cancer in the absence of BRCA1 and BRCA2 mutations, but because of a shared pathway, the same CHEK2 variants in the presence of high risk BRCA variants do not further increase risk [14], [24], [25].

Under each of the above scenarios, phenotype-genotype association testing is performed for rare NS variants. It is demonstrated that the KBAC has a clear advantage in power and robustness over other existing methods and this benefit is especially strong, when rare variant data is analyzed where there is either variant misclassification or gene interactions.

In order to further illustrate applications of the KBAC and other statistical methods, i.e., WSS, CMC, and RVE to carry-out association studies, energy metabolism traits and rare variants in ANGPTL 3, 4, 5 and 6 genes obtained from sequence data were analyzed. In addition to identifying the originally reported association between triglyceride levels and ANGPTL 4, KBAC identified associations for a.) body mass index and ANGPTL 5, b.) diastolic blood pressure with ANGPTL 6, c.) high density lipoprotein with ANGPTL 4, d.) triglyceride levels with ANGPTL 3 e.) very low density lipoprotein with ANGPTL 3 and ANGPTL 4.

Results

The results presented focus on simulations using simulated SFS from AA sequence data. Similar results are found for simulations using simulated SFS for EA and estimated SFS for AA and EA (Text S1 and Figures S2, S3, S4, S5, S6, S7, S8.). Although the power varies dependent on the underlying model used to generate the data, in all cases the KBAC is the most powerful method followed by the WSS, CMC and then the RVE.

Rare Variant Frequency Distributions in Generated Case-Control Samples

Rare NS variants carrier information is summarized (Table 1) for replicates used in power comparisons in the presence of misclassifications. Under the phenotypic model with variable genetic effects, when all variants (both non-causal and causal variants) were analyzed, 5.5% of cases and 3.4% of controls are carriers, with carrier frequency in cases 61% higher than in controls. When only causal variants are included, the fractions of carriers in cases and in controls are 3.8% and 1.7% respectively. The case rare variant frequency is approximately 2.3 times of the controls frequency, which implicates that average ORs of uncovered rare variants lie between 2 to 3. For the phenotypic model with fixed genetic effects, the results are similar. The carrier frequency observed in cases is around 2.5 times the frequency in controls. Compared to the model with fixed effects, lower frequency rare causal variants have larger ORs for variable effects model. The probability that these low frequency rare variants are uncovered in a case-control sample is higher. Therefore, in all scenarios examined, more rare variants sites are uncovered for the model with variable effects. When all the variants are included, 11% more rare NS variants sites are uncovered for the model with variable effects. The number of rare variants sites that are exclusive to cases or controls is also higher under the variable effect model. For example, when 100% of the variant sites are included in the analysis, 47.4% and 41.1% of the sites are found exclusively in either cases or controls for the variable and the fixed effects model, respectively. For both models, within a single gene, very few cases and controls carry more than one rare variant.

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Table 1. Rare variant summary statistics.

https://doi.org/10.1371/journal.pgen.1001156.t001

For the within gene interaction model (Table 2), similar patterns of NS variants sites and carrier frequencies are observed. When 100% of the rare variants are causal, 5.5% of the cases and 3.2% of the controls are carriers on average for a case-control sample. Due to interaction, frequency differences between cases and controls are mitigated. In the between gene interaction model (Table 2), higher case carrier frequency and more rare variants sites are observed for the high risk gene than for the low risk gene. The proportions of rare variants carriers for the two genes combined can be high, e.g. when 100% of the variants are causal, up to 12% of the cases can be rare variant carriers. Rare variants distributions can be found in the (Text S1) for main effects models (Table S1) and within and between gene interactions models (Table S2) using simulated SFS for EA, and for main effects models using estimated SFS for AA (Table S3) and EA (Table S4) with re-sequencing data from ANGPTL3, 4, 5, and 6 genes.

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Table 2. Rare variant summary statistics.

https://doi.org/10.1371/journal.pgen.1001156.t002

Evaluation of Type I Error

When permutation was used to evaluate significance for the KBAC, type I error was well controlled, because p-values were obtained empirically. Additionally, in order to ensure that the type I error for RVE is well controlled permutation is also used to obtain empirical p-values. For the WSS [21], CMC [20] method, it was previously demonstrated that for the analysis of rare variants, their type I errors are well controlled [20]. For moderate sample sizes e.g. 400 cases/400 controls, the distributions of p-values for the Monte Carlo approximation are very close to those obtained using permutations and theoretical expectations (Figure 1) and additionally type I error is well controlled.

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Figure 1. Quantile-Quantile (QQ) plot of p-values obtained from Monte Carlo approximation (left panel), permutation (right panel), and theoretical expectations.

P-values were estimated using 10,000 iterations and 10,000 permutations for Monte Carlo approximation and permutation, respectively. Four sample sizes were investigated: 200 cases/200 controls; 300 cases/300 controls, 400 cases/400 controls, and 500 cases/500 controls. A total of 3,000 replicates were used to generate the QQ plot for each sample size.

https://doi.org/10.1371/journal.pgen.1001156.g001

Power Comparison

Main effects model without misclassification.

For main effects model with fixed genetic effects and no misclassification (Figure 2), the power for KBAC, WSS CMC and RVE are respectively given by 82.5%, 77.7%, 73.9% and 14.8%. The power for RVE is much lower than the power for the other three methods. For the main effects model with variable genetic effects (Figure 3), the power for the four methods is given by 83.1%, 78.8%, 74.2% and 44.8%. The power of the RVE improves for the variable genetic effects model compared to the fixed genetics effect model; while the power for the other methods remains relatively unchanged. KBAC is consistently more powerful than WSS, CMC and RVE, e.g. for fixed effect model, KBAC is 6.1% more powerful than WSS, 11.6% more powerful than CMC, and 457.4% more powerful than RVE.

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Figure 2. Impact of misclassifications under main effects model with fixed genetic effects using simulated SFS for AA.

Each causal rare variant has an OR = 3.0. Power comparisons were made for the KBAC, WSS, CMC, and RVE when 0%∼60% of causal rare variants are excluded from the analysis (left panel) and when 0%∼100% of non-causal rare variants are included (right panel). A sample size of 1000 cases and 1000 controls was used for each scenario. P-values were empirically estimated using 5,000 permutations and power was evaluated for a significance level of using 2,000 replicates for each scenario.

https://doi.org/10.1371/journal.pgen.1001156.g002

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Figure 3. Impact of misclassifications under main effects model with variable genetic effects using simulated SFS for AA.

The disease odds for causal variants are inversely correlated with their MAFs and within the range of 2∼20. Power comparisons were made for the KBAC, WSS, CMC, and RVE when 0%∼60% of causal rare variants are excluded from the analysis (left panel) and when 0%∼100% of non-causal rare variants are included (right panel). A sample size of 1000 cases and 1000 controls was used for each scenario. P-values were empirically estimated using 5,000 permutations and power was evaluated for a significance level of using 2,000 replicates for each scenario.

https://doi.org/10.1371/journal.pgen.1001156.g003

Impact of misclassification.

Under both models (Figure 2, Figure 3), the power of all methods is negatively impacted by exclusions of causal variants and inclusions of non-causal variants at a varying degree. When non-causal variants are included in the analysis, KBAC is consistently more powerful and more robust than the other three methods. For example, when 100% of the non-causal variants are included, under the variable effects model, KBAC is 19.3% more powerful than WSS , 27.6% more powerful than CMC , and 91.0% more powerful than RVE . When compared under the fixed effects model, the advantage of KBAC over WSS , CMC and RVE remains largely unchanged. For the scenarios where causal variants are missing, the relative performances of the methods remain to be in the order KBAC>WSS>CMC>RVE. For the variable effects model, the power advantage of WSS over CMC is greater than the advantage observed for the fixed effects model. For example, when 60% of the causal variants are excluded from the analysis, under the fixed effects model, the power for WSS drops 40.1% and the power of CMC drops 45.1%, while under the variable effects model, the power decreases for WSS and CMC are respectively 39.1%, 47.8%. The KBAC is more robust than the other methods: the power decreases under the fixed and variable effects models are respectively 34.1% and 35.6%, which are smaller than the decreases in power for WSS and CMC. Exclusion of causal variants from the analysis is more detrimental to power than inclusion of non-causal variants. Power comparisons with simulated SFS for EA can be found in (Figure S2) for fixed effects model and in (Figure S3) for variable effects model. Additionally, power comparisons with estimated SFS for AA are shown in (Figure S4) for fixed effects and in (Figure S5) variable effects models and that with estimated SFS for EA are displayed in (Figure S6) for fixed and in (Figure S7) for variable genetic effects models.

Within gene interaction model.

Under the within gene interaction model, KBAC is consistently the most powerful method for all scenarios with different proportions of causal variants (Figure 4). The advantage of KBAC in the presence of interactions is apparent and its advantage over other methods becomes greater with increasing proportion of non-causal variants. For example, when all variants are causal, the power of KBAC is 8.4% higher than WSS, which is the second most powerful method. But when only 50% of all variants are causal, KBAC is 30.7% more powerful than WSS. RVE is the least powerful methods for all scenarios compared.

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Figure 4. Power comparisons for within gene (left panel) and between gene interaction model (right panel) with simulated SFS for AA.

Power was evaluated for the KBAC, WSS, CMC and RVE. A sample size of 1000 cases and 1000 controls were used for the within interaction model, and a sample size of 300 cases and 300 controls were used for the between gene interaction model. Scenarios with different proportions of causal variants were considered. P-values were empirically estimated using 5,000 permutations and power was evaluated for a significance level of using 2,000 replicates.

https://doi.org/10.1371/journal.pgen.1001156.g004

Between gene interaction model.

In the between gene interaction model, power comparisons between the four methods remain similar (Figure 4). KBAC is consistently the most powerful method and is robust against inclusion of non-causal rare variant sites. Comparing the scenario where all variants are causal with the scenario where only 50% of the variants are causal, the power for KBAC drops 36.3%, while the power for WSS drops 48.2%. Power comparisons for within and between gene interaction models using simulated SFS based on sequence data from EA can be found in (Figure S8).

ANGPTL Variants and Energy Metabolism in Humans

In order to further illustrate the application of KBAC and other rare variant analysis methods (i.e. WSS, CMC and RVE), rare variants in the ANGTPL 3,4,5 and 6 genes were analyzed to determine whether they are associated with energy metabolism traits (Table 3). As in the original DHS study [26], the association of rare variants in the ANGPTL3,4,5 and 6 genes with triglyceride (TG), low density lipoprotein (LDL), very low density lipoprotein (VLDL), high density lipoprotein (HDL), cholesterol, glucose, body mass index (BMI), systolic (SysBP) and diastolic blood pressure (DiasBP) were investigated. In the original DHS study, NS variants were analyzed using RVE, and significant associations were found between ANGPTL3, ANGPTL 4 and TG as well as between ANGPTL 6 and cholesterol [5], [6]. In this article NS variants, most of which are very rare [5], [6], were analyzed. Individuals with confounding factors (lipid lowering drugs, diabetes mellitus and heavy alcohol use) were removed for all analyses. Multiple associations were identified with KBAC but not with other approaches, i.e. the novel associations between ANGPTL 6 and DiaBP , as well as between ANGPTL 3 and TG levels . Additionally multiple novel associations were observed for analyses carried out with KBAC, WSS and CMC: 1.) ANGPTL4 and VLDL ; 2.) ANGPTL5 and BMI ; 3.) ANGPTL4 and HDL and 4.) the previously reported association between ANGPTL4 and TG levels . It should be noted that HDL and TG levels are negatively correlated (−0.42) and individuals with HDL levels in the lower quartile had an excess of rare variants in ANGPTL4 compared to those individuals with HDL levels in the upper quartile, while those individuals with TG levels in the upper quartile had an excess of rare variants in ANGPTL4 compared to those with TG levels in the lower quartile. The association detected by KBAC between ANGPTL4 and VLDL and between ANGPTL5 and BMI remains significant after correcting for multiple testing. RVE, on the other hand, detected associations between ANGPTL 5, 6 and glucose while the other three methods did not. We further investigated this association by applying a more stringent MAF cutoff 0.1% for the NS variants analyzed in ANGPTL 5 and 6. Using this new criterion both associations were detected by all methods (for ANGPTL 5, and for ANGPTL 6, ).

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Table 3. Association analyses of the ANGPTL 3,4,5 and 6 gene variants with human energy metabolism phenotypes.

https://doi.org/10.1371/journal.pgen.1001156.t003

Discussion

The KBAC method developed for association mapping of rare variants combines genotype classification and hypothesis testing in a coherent framework. The risk of each multi-site genotype is modeled as a mixture distribution with two components, among which only the component representing a non-causal genotype is known and is used in the adaptive weighting. Each multi-site genotype is continuously weighted using the non-causal component. The power of the KBAC as well as the other methods investigated can be affected by inclusion of non-causal mutations or exclusion of causal variants in the sample, to a varying degree. When non-causal variants are included in the analysis, the difference in rare variant carrier frequencies observed between cases and controls is mitigated. On the other hand, when causal variants are excluded from the association analysis, the marginal effect size of existing variants can vary considerably depending on whether missing causal variants exist on the same multi-site genotype. As a result, treating each variant (or multi-site genotype) interchangeably will incur loss of power, the severity of which will depend on the proportion of misclassified variants in the data. The performance of the KBAC is superior to the other approaches that were examined.

Bioinformatics tools [17], [18] and filters [1] can be used to determine which rare variants are potentially functional and should be included in the association analysis [1]. Their predictive accuracy, which can be low, is dependent on the amount of information available for the gene understudy. If bioinformatics tools are used to predict variant functionality and determine which variants should be included in the analysis it is best to loosen stringency, because the exclusion of causal variants is more detrimental to power than inclusion of non-causal variants. Whether or not bioinformatics tools are used as a screening tool, misclassification will occur therefore the robustness of KBAC to misclassification is particularly beneficial. Additionally in order to avoid potentially erroneous exclusion of causal variants due to locus heterogeneity, joint analysis of multiple putative genetic loci that carry similar functions or reside in the same pathway can be valuable.

It is of great interest to evaluate gene×gene interactions in the study of complex diseases. The KBAC analyzes multi-site genotypes (or multi-locus genotype), which can be beneficial in detecting gene interactions [11]. This property is especially important when multiple genetic loci are jointly analyzed in order to aggregate rare variants. Interactions are more likely to occur between genes involved in the same pathways. In addition, it has been hypothesized that functions of rare variants can be modulated by common variants [8]. Since the KBAC uses adaptive weighting instead of a fixed model, unknown patterns of gene interaction can be automatically integrated into the analysis. Through models motivated by Hirschsprung's disease and breast cancer, it is shown that in the presence of interactions the KBAC outperforms other approaches. An additional advantage of the KBAC is that kernel weights computed for adaptive weighting provide a measure with which the relative risk of each multi-site genotype can be assessed, for further replication studies.

The RVE method which compares the occurrence of variants which are exclusively observed in cases to those which are only observed in controls has the lowest power among all tests evaluated. The RVE method possesses undesired statistical properties by excluding those variants which are observed in both cases and controls. For all variants that are not fully penetrant, when sample size is large, they tend to appear in both case and control samples and would thus be excluded from the analysis using RVE. As a result, the RVE method is not asymptotically consistent; with increasing sample size power may be even lower than for smaller sample sizes [27].

Forward time simulations of locus genetic data incorporated both population demographic change and purifying selection. Both factors are known to impact SFS for observed rare variants (especially NS variants). Only NS variants were analyzed for comparing different methods, as it has been suggested that using NS variants will concentrate variations on functionally significant class of alleles, and increase signal to noise ratio [27]. There have been a number of studies on complex diseases which identified associations with NS variants [3], [5], [6]. When synonymous mutations are also considered in the analyses, higher proportions of non-causal variants may be introduced, so the adaptive property and the robustness of KBAC will be more advantageous.

Whether or not phenotypic effects of causal rare variants are inversely correlated with their MAF is unknown. Deleterious functional variants tend to have low frequencies [28], but the functional effect of a deleterious mutation may not be associated with the disease. On the other hand, for mutations involved in complex traits, they may not be at selective disadvantage due to the fact that most complex traits are late on-set and may not cause reductions in reproductive fitness. For both types of models, the advantage of KBAC is apparent. WSS and RVE perform better under the variable effects models, when only causal variants are present. This is because high risk causal variants are assigned higher weights. However, as low frequency non-causal variants also receive larger weights that negatively affect power, there are no measurable improvements of WSS compared to the model with fixed genetic effects. On the other hand, due to the adaptive nature of KBAC, the method performs consistently the best under both classes of models.

The KBAC test statistic does not have a closed form distribution; therefore it is necessary to evaluate significance either through permutation or using Monte Carlo approximation. For small sample sizes i.e. ∼≤400 cases and 400 controls, permutation is recommended, because it can be more reliable than Monte Carlo approximation. For larger sample sizes, Monte Carlo approximation not only controls type I error, but also the estimates of power do not differ from those obtained using permutations (data not shown). Permutation can be computationally intensive for large samples and/or genome-wide data where a large number of genetic regions are analyzed; therefore Monte Carlo approximation can be particularly advantageous to evaluate significance due to its computational efficiency.

A well known problem of genetic association studies is spurious findings due to population substructure and/or population admixture. For rare variant association analysis this problem can occur when study subjects are sampled from different populations and the distribution of non-causal variant sites and/or aggregate frequencies of non-causal variants differ between the sampled populations. To control for population stratifications, KBAC can be coupled with principal components analysis (PCA) [29] approach and eigenvector(s) can be included as covariates in the analysis (see Methods: Controlling for Confounders). PCA approach has been shown to be a powerful tool to accurately infer geographical locations [30], [31]. In addition, KBAC can also be used with clustering/matching based methods, such as structured association [32], [33] to control for population stratification.

The application of KBAC as well as WSS, CMC and RVE were further illustrated by the analyses of genes in ANGPTL family. In the analyses, all individuals with potentially confounding factors i.e. diabetics, alcoholics, and individuals treated with lipid lowering drug were excluded. In the original studies individuals were excluded based upon both their quantitative trait values and the confounding factors. For example, only individuals treated with lipids lowering drugs in the lower quartile of TGs were removed, but those in the upper quartile were included in the analysis. We believe excluding individuals based upon their quantitative trait values should not be done instead all individuals meeting the exclusion criteria should be removed from the analysis. KBAC performs consistently well, and identifies the most phenotype-genotype associations among all the approaches compared. The effects of mutant ANGPTL genes on lipoprotein lipase (LPL) have been studied through in vitro functional studies and in vivo mice studies. LPL has been known to affect glucose metabolism [34], cholesterol level [34]–[37], and blood pressure [38]. This biological evidence strengthens the support of the identified associations. Additionally, the association between variants in ANGPTL4 gene and triglyceride levels were successfully replicated using an independent dataset [5], [6].

Although the examples given are for the analysis of single regions and interaction between two regions, the KBAC can also be used to analyze entire exomes (or genomes). In order to control for family-wise error rate (FWER), it is sufficient to use a Bonferroni correction, since there will be little or no linkage disequilibrium between rare variants in different genes. It is thus not necessary to control the FWER using permutations. If exome sequencing is carried out and analysis is implemented gene by gene, given that human genome contains ∼20,000 genes, a significance level can be applied. The correction necessary for gene based association mapping of rare variants is less than the threshold currently used for genome-wide association studies [39] which is usually .

The KBAC is a powerful tool to detect main association effects and gene interactions in large sequence data sets of candidate genes, exomes and in the future entire genomes. The KBAC is implemented in a user friendly R package and is available from the authors.

Methods

Supporting Information

Acknowledgments

We would like to thank Drs. Jonathan Cohen and Helen Hobbs for providing us with data from the Dallas Heart Study on the ANGTPL family genes. Additionally we would like to thank Drs. William Amos, Hua Chen, Andrew DeWan, Marek Kimmel, Bingshan Li, Fuli Yu, and Andreas Ziegler for useful conversations and suggestions.