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With Rare Variants

Saonli Basu and Wei Pan

Division of Biostatistics, School of Public Health, University of Minnesota, Minneapolis, Minnesota

In anticipation of the availability of next-generation sequencing data, there is increasing interest in investigating association

between complex traits and rare variants (RVs). In contrast to association studies for common variants (CVs), due to the low

frequencies of RVs, common wisdom suggests that existing statistical tests for CVs might not work, motivating the recent

development of several new tests for analyzing RVs, most of which are based on the idea of pooling/collapsing RVs.

However, there is a lack of evaluations of, and thus guidance on the use of, existing tests. Here we provide a comprehensive

comparison of various statistical tests using simulated data. We consider both independent and correlated rare mutations,

and representative tests for both CVs and RVs. As expected, if there are no or few non-causal (i.e. neutral or non-associated)

RVs in a locus of interest while the effects of causal RVs on the trait are all (or mostly) in the same direction (i.e. either

protective or deleterious, but not both), then the simple pooled association tests (without selecting RVs and their association

directions) and a new test called kernel-based adaptive clustering (KBAC) perform similarly and are most powerful; KBAC

is more robust than simple pooled association tests in the presence of non-causal RVs; however, as the number of non-causal

CVs increases and/or in the presence of opposite association directions, the winners are two methods originally proposed

for CVs and a new test called C-alpha test proposed for RVs, each of which can be regarded as testing on a variance

component in a random-effects model. Interestingly, several methods based on sequential model selection (i.e. selecting

causal RVs and their association directions), including two new methods proposed here, perform robustly and often have

statistical power between those of the above two classes. Genet. Epidemiol. 35:606–619, 2011. r 2011 Wiley Periodicals, Inc.

Key words: C-alpha test; kernel machine regression; logistic regression; model selection; permutation; pooled association

tests; random-effects models; SSU test; Sum test; statistical power

Contract grant sponsor: NIH; Contract grant numbers: R21DK089351; R01HL65462; R01HL105397.

Correspondence to: Wei Pan, Division of Biostatistics, MMC 303, School of Public Health, University of Minnesota, Minneapolis,

MN 55455--0392. E-mail: weip@biostat.umn.edu

Received 30 November 2010; Revised 23 March 2011; Accepted 3 June 2011

Published online 18 July 2011 in Wiley Online Library (wileyonlinelibrary.com/journal/gepi).

DOI: 10.1002/gepi.20609

INTRODUCTION statistical test for GWAS based on testing single SNVs is

not expected to perform well. In fact, in light of the

Genome-wide association studies (GWASs) have success- significant difference in variant frequencies between RVs

fully identified thousands of common genetic variants, and CVs, common wisdom might suggest that many

mainly common single nucleotide variants (SNVs), asso- existing methods for CVs would not work either, motivat-

ciated with complex traits, including many common ing the development of new statistical tests specifically

diseases [Hindorff et al., 2010]. However, these identified targeting RVs. The most striking feature of several recently

variants can only explain a small proportion of inheritable proposed new tests for RVs is the idea of pooling or

phenotypic variance [Maher, 2008], leaving the door open collapsing: rather than testing on individual SNVs one by

for many more yet to be discovered variants. A popular one (as in GWASs), one would pool or collapse multiple

hypothesis is that many more rare variants (RVs) may rare SNVs together such that collectively they would have

contribute to the missing heretability unexplained by a reasonably high frequency, and then apply a test to the

discovered common variants (CVs) [Bodmer and Bonilla, collapsed genotype [Morgenthaler and Thilly, 2007; Li and

2008; Gorlov et al., 2008; Pritchard, 2001; Pritchard and Cox, Leal, 2008; Madsen and Browning, 2009; Price et al., 2010].

2002]. At the same time, biotechnological advances have Albeit well motivated and shown to perform better than

made it feasible to re-sequence parts of or whole genomes. single SNV-based testing, such a pooling strategy has its

In anticipation of the arrival of massive amounts of next- own limitations. If the RVs to be pooled are associated

generation sequencing data, the chance of success in with the trait in different directions, i.e. some are

detecting association between complex traits and RVs associated positively while others negatively, the strategy

largely depends on statistical analysis strategies for RVs; of pooling may weaken or diminish the signal in

see two excellent timely reviews [Asimit and Zeggini, associated RVs. Furthermore, if many of the RVs are

2010; Bansal et al., 2010]. Since frequencies of RVs are very non-causal, i.e. they are not associated with the traits,

low, even with high penetrance, it will be difficult to detect pooling will inevitably introduce noises into the collapsed

Statistical Tests for Rare Variants 607

genotype and thus have reduced statistical power. Note coded by an additive genetic model: Xij 5 0, 1 or 2 for the

that, the effects of RVs are not always in the same number of the RV (minor allele) for SNV j, j ¼ 1; . . . ; k.

direction: they can be protective or deleterious. For

example, some RVs in gene PCSK9 are associated with METHODS ORIGINALLY PROPOSED FOR CVS

lower plasma levels of low-density lipoprotein cholesterol

(LDL-C) while others associated with higher levels of Logistic regression. Several most popular statistical

LDL-C [Kotowski et al., 2006]. In recognition of these tests are based on logistic regression:

limitations, several methods based on model selection X

k

have been proposed recently [Han and Pan, 2010a; Logit PrðYi ¼ 1Þ ¼ b0 1 Xij bj : ð1Þ

Hoffmann et al., 2010; Bhatia et al., 2010; Zhang et al., j¼1

2010]. The main idea is to determine whether a RV should

be pooled, and if so, what is its association direction. Since The null hypothesis to be tested is H0: b¼ðb1 ; . . . ; bk Þ0 ¼0.

these methods are based on either a marginal test or a step- Maximum likelihood can be utilized to derive asymptoti-

up procedure on each individual RV, the power of cally equivalent score test, Wald’s test and likelihood ratio

selecting a RV and determining its association direction test (LRT); here we focus on the score test for its

may be limited. Here we propose two new model-selection computational simplicity. For model (1), the score vector

procedures that improve over the existing pooled associa- and its covariance matrix are

tion tests while maintaining low computational cost, X

n

borrowing the idea of Basu et al. [2010] in linkage analysis. U¼ i;

ðYi YÞX

Very recently several new tests, including a kernel-based i¼1

adaptive clustering (KBAC) [Liu and Leal, 2010], a C-alpha X

n

test [Neale et al., 2011] and a replication-based test (RBT) YÞ

V ¼ Yð1

ðXi XÞðX 0

i XÞ ;

[Ionita-Laza et al., 2011], specifically designed for RVs and i¼1

aiming to overcome various weaknesses of the pooled Pn Yi =n and X

where Xi ¼ðXi1 ; . . . ; Xik Þ0 , Y¼ ¼ Pn Xi =n.

association tests, have appeared. However, no comparison i¼1 i¼1

The most popular test for CVs in GWAS is the

was made among these new tests and model-selection

(univariate) minP (UminP) method that tests on each

approaches for RVs. More generally, in the current

single SNVs one-by-one and then takes the minimum of

literature, there is no evaluation on the applicability of

their P-values. The corresponding UminP score test

most existing tests to RVs. Although most existing tests

statistic is

have been proposed for and mainly applied to CVs, some

were originally developed for high-dimensional data and TU min P ¼ max Uj2 =Vjj ;

thus are likely to be robust to the large number of j¼1;...;k

parameters facing the analysis of RVs, and may have where Uj is the jth element of U and Vjj is the (j,j) th

reasonable power for RVs. Goeman’s score test [Goeman element of V. An adjustment for multiple testing has to be

et al., 2006] and kernel machine regression (KMR) [Liu et al., made. Although the Bonferroni and permutation methods

2008] are two such examples. Since Goeman’s test is are most commonly used, a better way is to derive the null

permutation-based and is equivalent to a test called the distribution of TU min P and thus a P-value based on

sum of squared score (SSU) test [Pan, 2009], we consider the numerical integration with respect to a multivariate

SSU test here. As to be shown, perhaps surprisingly, both Gaussian density [Conneely and Boehnke, 2007].

the SSU test and KMR, along with the C-alpha test A joint test as an alternative to the UminP test is the

specifically proposed for RVs [Neale et al., 2011], performed multivariate score test:

extremely well under certain situations when the pooled

association tests had low power. In summary, given the TScore ¼ U 0 V1 U;

compelling interest of the scientific community in detecting

association between complex traits and RVs while little is which has an asymptotic chi-squared distribution with

known about the relative performance and merits of degrees of freedom (DF) k. If DF k is large, the test may not

various existing and new tests, it is timely to have a have high power.

comparative evaluation of the tests, an endeavor taken here. Pan [2009] proposed two tests, called SSU and sum of

weighted squared score (SSUw) tests:

where Diag(V) is a diagonal matrix with the diagonal

To be concrete, we restrict the attention to the case- elements of V. Under H0, each of the two test statistics has

control design with a binary trait, say disease, though an asymptotic distribution of a mixture of w21 ’s, which can

many of the methods discussed are based on logistic be approximated by a scaled and shifted chi-squared

regression and can be easily extended to generalized linear distributions [Pan, 2009]. The two tests can be regarded as

models (GLMs) for other types of traits. We do not modified score test by ignoring the non-diagonal elements

consider adjusting for covariates, such as environmental of V, i.e. correlations among the components of U, which is

factors, though again methods based on logistic regression known to be advantageous for high-dimensional data

can easily accommodate covariates. We assume that the [Chen and Qin, 2010]. More importantly, as shown by Pan

analysis goal is to detect whether there is any association [2009], the SSU test is equivalent to the permutation-based

between the disease and a group of rare SNVs, for version of [Goeman et al., 2006] which is derived as a

example, SNVs in a sliding window or in a functional variance component score test for a random-effects (R-E)

unit such as gene. We denote the binary trait Yi 5 0 for n0 logistic regression model. Specifically, in model (1), if we

controls, and Yi 5 1 for n1 5 nn0 cases. The k variants are assume bj’s as random effects drawn from a distribution

Genet. Epidemiol.

608 Basu and Pan

with E(b) 5 0 and CovðbÞ ¼ t, then Goeman’s score test difference between the SSU and SSUw tests, illustrating

on H0: t 5 0 is when one of the two is more powerful than the other. For

example, if the causal variants tend to have lower MAFs

S ¼ 12U 0 U 12trðVÞ; ð2Þ than that of non-causal ones, the SSU test is expected to be

less powerful; otherwise, the SSU test is more powerful.

where tr(A) is the trace of matrix A. Observing that V is A potential problem with the above weighting scheme

invariant to permutations of Y, we know that, under (and with the SSUw test) is that, since a causal variant may

permutations, using S is equivalent to using SP ¼ U 0 U, have a higher MAF in cases but a lower MAF in controls,

which is equivalent to the SSU, SSUw and score test and thus a higher overall MAF across both cases and

statistics with ¼ I, ¼ DiagðVÞ and ¼ V, respectively. controls, it will downweight this causal variant, leading to

Note that Goeman’s test was originally derived to test on a reduced power. This is a reason that Madsen and

large number of parameters for high-dimensional micro- Browning [2009] proposed using the MAFs of only

array data, though its good performance for lower-

controls to construct weights. Specifically, if there are n0j

dimensional SNV data have been empirically confirmed

too [Chapman and Whittaker, 2008; Pan, 2009]. minor alleles for variant j in all the controls, then we can

Another test performed well under certain situations for use weights

CVs is the so-called Sum test, as noted by Chapman and qﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

Whittaker [2008] and Pan [2009]. The Sum test was wj ¼ 1= nqi ð1 qj Þ; qj ¼ ðn0j 11Þ=ð2n0 12Þ: ð4Þ

motivated to strike a balance between jointly testing on

multiple SNVs and its resulting DF. The Sum test is based With such weights, which already use the disease labels,

on a key and generally incorrect working assumption that the asymptotic SSU test (and Sum test) would have

the SNVs are all associated with the trait with a common inflated Type I error rates. Alternatively, we use a standard

association strength: permutation to calculate P-values, and denote the result-

ing test wSSU-P.

X

k Logistic KMR and genomic similarity-based

Logit PrðYi ¼ 1Þ ¼ bc;0 1 Xij bc ; ð3Þ methods. Rather than testing the effects of the SNVs

j¼1 parametrically (i.e. linearly in our specified model (1)), one

can adopt a nonparametric model:

where bc reflects the common odds ratio (OR) between the

trait and each SNV under the working assumption. While Logit PrðYi ¼ 1Þ ¼ b0 1hðXi1 ; . . . ; Xik Þ; ð5Þ

utilizing all the SNVs, the Sum test avoids the possibly too

where h( ) is an unknown nonparametric function to be

large DF, and thus loss of power, of other multivariate

estimated, offering the flexibility in modeling the effects of

tests. It only requires to test on a single parameter with H0:

the SNVs on the trait. In a specific approach called KMR

bc 5 0 by a score test (or its asymptotically equivalent

[Liu et al., 2008], the form of h( ) is determined by a user-

Wald’s test or LRT). Pan [2009] pointed out that the

specified positive and semi-definite (psd) kernel function

weighted score test of Wang and Elston [2007] share the

KðXi :; Xj Þ, which measures the genomic similarity between

same spirit and thus similar performance as the Sum test.

Note that in model (3) we regress Y on a new ‘‘super-SNV’’ the genotypes of subjects i and j. Some commonly used

that is the sum of the genotype values of all the SNVs, kernels include linear, identity-by-descent (IBS) and quad-

hence we call the resulting test Sum test. ratic kernels. By the representer P theorem [Kimeldorf and

To incorporate prior biological information, one may Wahba, 1971], hi ¼ hðXi Þ ¼ nj¼1 gj KðXi ; Xj Þ with some

want to weight SNVs using some suitable weights, e.g. g1 ; . . . ; gn . To test the null hypothesis of no association

based on their MAFs [Madsen and Browning, 2009] or between the phenotype and SNVs, one can test H0:

their predicted likelihoods of being functional [Price et al., h ¼ ðh1 ðX1 Þ; . . . ; hn ðXn ÞÞ0 ¼ 0. Denote K as the n n matrix

2010]. It is straightforward to do so in logistic regression: with the (i,j) th element as KðXi ; Xj Þ and g ¼ ðg1 ; . . . ; gn Þ0 ,

with a set of weights w ¼ ðw1 ; . . . ; wk Þ0 , we can simply then we have h 5 Kg. Treating h as subject-specific random

weight the codings for SNVs; that is, rather than effects with mean 0 and covariance matrix tK, testing H0:

using Xi ¼ ðXi1 ; . . . ; Xik Þ0 for subject i, we use Xi;w ¼ h 5 0 for no SNV effects is equivalent to testing H0: t 5 0.

ðw1 Xi1 ; . . . ; wk Xik Þ0 in logistic regression model (1). It is The corresponding variance component score test statistic is

easy to see that, the UminP, score and SSUw tests are

0 KðY Y1Þ;

Q ¼ ðY Y1Þ

invariant to such weighting, while the SSU and Sum tests

do depend on such weighting. In fact, a careful examina- whose asymptotic null distribution is a mixture of w21 ’s,

tion of the SSU and Sum test statistics indicates that the which can be approximated by a scaled chi-squared

above two tests treat Xij’s more or less equally across j. By distributions [Wu et al., 2010].

the expression of V, we see that those variants with larger The above logistic KMR can be extended to include

MAFs tend to have larger variances for their components other covariates and for other traits, e.g. linear models for

of the score vector. Hence, without weighting, the SSU and quantitative traits [Kwee et al., 2008]. Since the kernel

Sum tests essentially give heavier weights to the variants function measures the similarity of two genotypes, KMR is

with larger MAFs, implying that they will be sensitive to expected to be related to genomic-distance based regres-

the presence of non-causal CVs, as to be confirmed. sion (GDBR) of Wessel and Schork [2006]; see Schaid

To overcome the above weakness of the SSU test, a [2010a,b] for a review on the topic. More specifically, as

simple strategy is to weight each variant j inversely by its shown by Pan [2011], both KMR and GDBR are equivalent

sample standard deviation SDðX1j ; . . . ; Xnj Þ, which is to the SSU test on H0: b 5 0 in a new logistic regression

equivalent to standardizing each predictor j to have a model:

sample SD 5 1. The resulting SSU test is essentially the

same as the SSUw test. This point highlights a key Logit PrðY ¼ 1Þ ¼ b0 1Zb; ð6Þ

Genet. Epidemiol.

Statistical Tests for Rare Variants 609

where K 5 ZZ0 . Hence, the difference between the SSU test the sums themselves, between the case and control groups.

for model (1) and logistic KMR is only in the transforma- Hence, putting aside the difference in weighting, the

tion of SNV codings in model (6), while both tests are wSum test is analogous to the Mann–Whitney–Wilcoxon

actually an SSU test applied to two different regression rank test, while other pooled association tests are

models. A special case is that, for a linear kernel K, we analogous to the t-test.

have K 5 XX0 ; that is, Z 5 X, under which the SSU and The main advantage of the above pooled association

KMR are equivalent. tests is their minimum DF at 1, hence no loss of power due

Empirically it has been found that GDBR and KMR to large DF or multiple testing adjustment. However, as

performed very well in detecting disease association with pointed out by Han and Pan [2010a], they all share a

CVs [Lin and Schaid, 2009; Wu et al., 2010; Han and Pan, common weakness: they suffer from possibly significant

2010b]. Albeit proposed for and mainly applied to CVs, power loss if the association directions of the causal

first Wessel and Schork [2006], and more recently Bansal variants are opposite. This can be most clearly seen from

et al. [2010] commented that GDBR (and thus KMR) could the Sum test. Generally, the common association para-

be applied to sequence data to detect association with RVs. meter bc in (3) can be viewed as a weighted average of the

To our knowledge, the above statistical tests originally individual b1 ; . . . ; bk ; see a closed-form expression for b^ c

proposed for CVs have never been applied to RVs. for linear regression given in Pan [2009]. Hence, depend-

Intuition might argue against their application to RVs. ing on the signs of b1 ; . . . ; bk , |bc| may be very small,

However, as to be shown, perhaps quite surprisingly, some leading to loss of power in the Sum test. To overcome this

of them performed quite well in our numerical studies. We limitation, several methods based on model selection have

will offer some explanations in Discussion. been proposed, as to be presented next.

Methods based on model selection. A general

METHODS FOR RVs model has been proposed by Hoffmann et al. [2010]:

Pooled association tests. The first test specifically X

k

designed for RVs is perhaps the cohort allelic sums test Logit PrðYi ¼ 1Þ ¼ bc0 1 gj Xij bc ; ð8Þ

(CAST) [Morgenthaler and Thilly, 2007]. CAST works by j¼1

first collapsing the genotypes across RVs to generate a

‘‘super-variant’’: Xi;C ¼ 1 if any Xij40 (i.e. any RV is with gj ¼ wj sj , where wj is a weight assigned to SNV j, sj 5 1

present), and Xi;C ¼ 0 otherwise. It then tests the associa- or 1 indicating whether the effect of SNV j is positive or

tion between the trait and this new Xi;C. It can be regarded negative, and sj 5 0 indicating the exclusion of SNV j from

as fitting a logistic regression model the model (i.e. the SNV is unlikely to be associated with the

trait). Madsen and Browning [2009] suggested to weight

Logit PrðYi ¼ 1Þ ¼ bC;0 1Xi;C bC ; ð7Þ RVs with the weights depending on their MAFs. However,

it is still debatable on how to appropriately weight the

and testing H0: bC 5 0. The most striking feature of CAST, SNVs, and if needed, it is not difficult to incorporate a

as the Sum test, is its testing on a single parameter, thus weighting scheme into most methods discussed here.

low DF and possibly increased power. Hence, we do not discuss the use of weights and always

As pointed out by Han and Pan [2010a], the CAST is assume wj 5 1 for any test except the wSum test.

closely related to the Sum test: both test on only a single The pooled association tests correspond to fixing sj 5 1

parameter representing some average effect of the multiple for all j’s. Several existing model-selection-based methods

SNVs. They differ in their coding of the ‘‘super-variant’’: can be classified into one of the two classes:

P

Xi;C ¼ _kj¼1 Xij versus Xi;S ¼ kj¼1 Xij , similar to the use of a (1) Choosing sj 5 1 or 1 in a data-dependent manner.

dominant genetic model versus an additive genetic model Han and Pan [2010a] proposed an adaptive Sum

for the effect of an individual variant. Note for RVs, we (aSum) test, in which the value of each sj is determined

have Xi;C Xi;S . Other codings for the ‘‘super-variant’’ are based on a univariate test on the marginal association

also possible, as considered by Morris and Zeggini [2010]. between the trait and SNV j for j ¼ 1; . . . ; k.

Li and Leal [2008] proposed a new test called Combined (2) Choosing sj 5 1, 0 or 1 in a data-dependent manner.

Multivariate and Collapsing (CMC) test, which modifies A Step-up procedure [Hoffmann et al., 2010] and a

the CAST to improve its performance when both rare and covering method (called RareCover) [Bhatia et al.,

CVs are present. Specifically, for any rare mutations with 2010] have been proposed to determine the value of

their minor allele frequencies (MAFs) less than some sj’s, both in a manner of forward variable selection:

threshold, say 0.05, they will be combined into a new starting from a null model without any SNV, SNVs are

group as in the CAST, while each CV (e.g. with selected one by one based on their statistical signifi-

MAF40.05) forms its own group, and the generalized cance and then added into the model.

Hotelling’s test [Fan and Knapp, 2003; Xiong et al., 2002] is

applied to such formed multiple groups. Note that the Here we propose two new methods, both of which start

generalized Hotelling’s test is closely related to the score from the Sum test with all sj 5 1. The main motivation is

test in logistic regression [Clayton et al., 2004]. Hence, for that, since the individual effect of each RV is hard to detect

only RVs, the CMC test is essentially the same as the CAST while the Sum test (or any other pooled association test)

(and the Sum test). has proven useful for RVs, rather than starting from a null

The weighted sum (w-Sum) test of Madsen and model (as in the Step-up and the RareCover procedures) or

Browning [2009] is also based on the idea of collapsing testing on marginal association (as in the aSum test), we

RVs. It differs from the Sum test in (i) using a weighted would like to start from the Sum test and make any

sum, instead of a simple sum, of RVs by their MAFs, and necessary adjustment on the values of sj’s, which may

(ii) comparing the ranks of the weighted sum, rather than result in higher power. In the first method, called

Genet. Epidemiol.

610 Basu and Pan

Sequential Sum test (Seq-Sum), for each SNV j with j 5 1 with C(a,b) as the combination number of choosing b out

and increased to k, we determine which of the two models, of a. The P-value is calculated by standard permutations

the current model with sj 5 1 and the other model with (for small samples while a Monte Carlo approximation is

sj 5 1 (while all other sj’s fixed at their current values for used for large samples).

both models), is preferred based on which model yields a From the expression of TKBAC , we see that its perfor-

larger (maximized) likelihood; then we increase j by one mance may deteriorate in the presence of both protective

and repeat the above process until we have tried and harmful causal variants: some positive and negative

j ¼ 1; . . . ; k. In the second method, called Seq-Sum test components ðn1;i =n1 n0;i =n0 Þwi may cancel out with each

with variable selection (Seq-Sum-VS), starting from SNV other in the sum, though the use of weight wi may alleviate

j 5 1, we consider three models with sj 5 1, 0 and 1, the problem. A simple modification as shown below may

respectively, and choose the model with the largest help overcome the problem:

(maximized) likelihood; then we increase j by 1, and

repeat the above process until having tried j ¼ 1; . . . ; k X

M

TmKBAC ¼ ðn1;i =n1 n0;i =n0 Þ2 wi ;

sequentially. Hence Seq-Sum considers only the coding of

i¼1

each SNV (i.e. its protective or harmful effect), while

Seq-Sum-VS considers selecting both SNVs and their though we do not pursue it here. In addition, KBAC

association directions. It is noted that the two methods includes non-causal variants in forming mutation patterns,

consider only a total of k11 and 2k11 candidate models which may dramatically increase the number of mutation

respectively. Due to the nature of their sequential search patterns (M) and thus effectively reduce the group sizes

and dependence on the order of the SNVs, unlike the n:i ’s, leading to loss of power. Nevertheless, the KBAC test

Step-up and CoverRare procedures, it is unlikely that they is attractive in detecting possible interactions among the

will select the best model (in terms of the largest variants, though we do not pursue this issue here.

maximized likelihood). Nevertheless, there are two possi- C-alpha test. Neale et al. [2011] proposed using the

ble benefits. One is the obviously reduced computational C-alpha test of Neyman and Scott [1966]. It is based on

cost when compared to an exhaustive search for exponen- testing for a common value (i.e. homogeneity) for a set of

tially many (i.e. 2k and 3k) models. The second benefit is binomial proportions, not on logistic regression.

less obvious: there is also lower cost for multiple testing For SNV j, assume there are nj subjects with the rare

adjustment due to a reduced number of model compar- mutation (or minor allele); among those nj subjects, we

isons. Computationally, rather than using the maximized have mj cases with mutation (and njmj controls with

likelihood as the criterion to select models, which requires mutation). We assume mjBin(nj,pj). Under the null

fitting each model by an iterative algorithm to obtain the hypothesis of no association between the disease and

maximum likelihood estimates, we adopt a score test, SNV j, we have pj 5 p0 for some common p0 for all

which is computationally much faster. The proposed j ¼ 1; . . . ; k. For a case-control study as considered here, we

Seq-Sum test is closely related to a new aSum test of Pan have p0 ¼ 1 n0 =n. The C-alpha test is based on the

and Shen [2011], which is more flexible while overcoming following:

a weakness of the Seq-Sum method, namely, its depen-

dence on an arbitrary ordering of the SNVs. X

k X

k

TC ¼ TC;j ¼ ðmj nj p0 Þ2 nj p0 ð1 p0 Þ;

In general, it is difficult to analytically derive the null j¼1 j¼1

distribution of a test statistic after model selection. For each

procedure above, we use permutations to calculate P-values. X

k X

k

VC ¼ VarðTC;j Þ ¼ E½ðmj nj p0 Þ2 nj p0 ð1 p0 Þ2 ;

Kernel-based adaptive clustering. Liu and Leal

j¼1 j¼1

[2010] proposed a method called KBAC for RV association

testing. KBAC works by grouping/clustering mutation where

patterns across the variants, and assigning each mutation nj

pattern a kernel-based weight adaptively determined by X

data. Specifically, suppose that among the cases and VarðTC;j Þ ¼ ½ðu nj p0 Þ2 nj p0 ð1 p0 Þ2 fðujnj ; p0 Þ

u¼0

controls, we have M11 mutation patterns across all k

variants, denoted as G0, G1 ; . . . ; GM , where G0 represents and fðujnj ; p0 Þ ¼ Cðnj ; uÞpu0 ð1 p0 Þnj u is the binomial prob-

the wild-type without any mutation. We also assume that ability PrðU ¼ uÞ for UBin(nj,p0). If all mj’s are independent,

there are n1;i cases and n0;i controls with mutation pattern then under the null hypothesis of no association p between

ﬃﬃﬃﬃﬃﬃ

Gi; denote n:i ¼ n1;i 1n0;i . For mutation pattern Gi, the risk any SNV and the disease, the test statistic Z ¼ TC = VC has

of having disease is estimated as Ri ¼ n1;i =n:i . The KBAC an asymptotic distribution of N(0,1), from which a P-value

test statistic is can be calculated. Alternatively, one can permute the

disease labels Y, calculate Z’s for permuted data and thus a

!2 P-value. We denote the two versions of the tests using the

X

M

TKBAC ¼ ðn1;i =n1 n0;i =n0 Þwi ; asymptotic distribution and the permutation distribution

i¼1

respectively as C-alpha-A and C-alpha-P.

The C-alpha test treats SNV-specific mutation rates pj’s

where the weight wi is determined by a hyper-geometric as a random sample drawn from some common distribu-

kernel: tion, say G. Under H0, the distribution reduces to a point

Z Ri mass at p0. Hence, the C-alpha test can be regarded as

X Cðn:i ; n:i rÞCðnn:i ; n1 n:i rÞ testing on the variance component of G: the variance of pj’s

wi ¼ k0i ðrÞ dr ¼ is 0 under H0. The C-alpha test is a score test for such a

0 n o Cðn; n1 Þ

r2 0 1

n:i ;n:i ;...Ri

homogeneity problem [Zelterman and Chen, 1988],

bearing some similarity to the framework of the variance

Genet. Epidemiol.

Statistical Tests for Rare Variants 611

component testing for a R-E model, under which the SSU Although the RBT was designed to differentiate between

and KMR can be formulated. In fact, as shown in Appendix, protective and harmful variants, it treats and tests the two

the general homogeneity score test of Zelterman and Chen groups separately, hence may lose power. Furthermore, for

[1988] has the same form of Goeman’s test. a non-causal variant j, it is likely that nj6¼mj/2, under

Each component of the C-alpha test statistic, TC;j , which case non-causal variant j will be pooled over into

contrasts the sample variance for variant j with its the test statistic, though its weight wj may be relatively

theoretical variance under H0. Since the fourth central small; nonetheless, the RBT may lose power in the

moment is presence of a large number of non-causal RVs.

A summary. We compare the above tests in several

Eðmj nj p0 Þ4 ¼ 3ðnj p0 q0 Þ2 1nj p0 q0 ð1 6p0 q0 Þ aspects as shown in Table I. We do not include CAST and

with q0 ¼ 1 p0 , under H0, we have CoverRare since they are similar to CMC and Step-up,

respectively. We note that the wSum test uses permuta-

VarðTC;j Þ ¼ 2ðnj p0 q0 Þ2 1nj p0 q0 ð1 6p0 q0 Þ; tions to estimate the mean and variance of its asymptotic

Normal distribution, and does not need a large number of

which is an increasing function of nj. Thus, similar to the permutations to reach high statistical significance, which is

SSU test (and KMR), since the C-alpha test statistic

P is a required by other permutation-based tests. We also note

simple sum of the statistics for the variants, TC ¼ kj¼1 TC;j , that the CMC was proposed to use the generalized

it may be dominated by the variants with large VarðTC;j Þ, Hotelling’s test, which does not accommodate covariates

e.g. those with high MAFs; it is possible, and even and other types of traits as shown in Table I. However,

productive, to weight the components suitably with since Hotelling’s test is equivalent to the score test in

a set of weights wj’s to yield a weighted version of the logistic regression [Clayton et al., 2004], it is easy to

C-alpha test: generalize the CMC test to accommodate covariates and

X

k other types of traits if the score test in a GLM is adopted.

TC;w ¼ wj TC;j : Finally, we will call the Sum, CMC and wSum tests loosely

j¼1 as the pooled association tests (that do not consider

selecting SNVs and their association directions).

As to be shown, similar to the SSU test, the C-alpha test

does not perform well in the presence of non-causal CVs,

in which case its weighted versions are more powerful. Weﬃ SIMULATED DATA

pﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ

can use weights wj as shown in (4), or wj ¼ 1= VarðTC;j Þ, We generated simulated data as in Wang and Elston

and calculate their -P-values using permutations; we [2007] and Pan [2009]. Specifically, we simulated k SNVs

denote the resulting tests as w1C-alpha-P and w2C- with the sample size of 500 cases and 500 controls. Each RV

alpha-P respectively. Since VarðTC;j Þ ¼ 0 and 0.25 for had a mutation rate or MAF uniformly distributed between

nj 5 1 and nj 5 2 respectively, we define wj 5 1/0.5 for 0.001 and 0.01, while for a CV it was between 0.01 and 0.1.

nj 5 1, the same as wj for nj 5 2, in the w2C-alpha-P test. As First, we generated a latent vector Z ¼ ðZ1 ; . . . ; Zk Þ0 from a

to be shown, the two weighted C-alpha tests did not multivariate normal distribution with a first-order auto-

perform as well as the wSSU-P test, and will be skipped in regressive (AR1) covariance structure: there was an

most of simulations. correlation CorrðZi ; Zj Þ ¼ rjijj between any two latent

Replication-based test. Ionita-Laza et al. [2011] components. We used r 5 0 and r 5 0.9 to generate

proposed a new test called RBT. The RBT is similar to a (neighboring) SNVs in linkage equilibrium and in linkage

pooled association test but purposefully designed to deal disequilibrium (LD) respectively. Second, the latent vector

with possibly different association directions. In addition, was dichotomized to yield a haplotype with MAFs each

a new weighting scheme is adopted to improve power. randomly selected. Third, we combined two independent

Using the same notation as before, suppose that for variant haplotypes and obtained genotype data Xi ¼ ðXi1 ; . . . ; Xik Þ0 .

j there are nj mutations in cases and mjnj mutations in Fourth, the disease status Yi of subject i was generated from

controls. Define a statistic to measure the enrichment of the logistic regression model (1). For the null case, we used

mutations in cases: b 5 0; for non-null cases, we randomly selected 8 non-zero

components of b while the remaining ones were all 0. Fifth,

X

k

as in any case-control design we sampled 500 cases and 500

S1 ¼ Iðnj 4mj =2Þwðnj ; mj Þ controls in each dataset.

j¼1

We considered several simulation set-ups. Throughout

with weight the simulations, we fixed the test significance level at

a 5 0.05 (or a 5 0.01 in a few cases), and used 500

wðnj ; mj Þ ¼ log Prðnj ; mj Þ permutations for each permutation-based method. The

¼ logðppoisðmj nj ; mj =2Þ½1 ppoisðnj 1; mj =2ÞÞ; results were based on 1,000 independent replicates for

each set-up.

where ppois(a,b) is the cumulative distribution function of a We used the R code of Wu et al. [2010] implementing the

Poisson distribution Pois(b) evaluated at a. Similarly we KMR methods. We used the linear, IBS and quadratic

measure the enrichment of mutations in controls with kernels; since the first two performed similarly across all

simulations, we present results for the linear and quadratic

X

k

kernels. We used the R package thgenetics implementing

S ¼ Iðnj omj =2Þwðmj nj ; mj Þ:

j¼1

the Step-up procedure, and a C11/R implementation of

KBAC. We implemented all other tests in R. For the CMC

The final test statistic is TR ¼ maxðS1 ; S Þ. The P-value is test, we used the default cut-off of MAF r0.05 for RVs,

calculated by permutations. though we explored using the cut-off r0.01 in a few cases.

Genet. Epidemiol.

612 Basu and Pan

TABLE I. A summary of the properties of the tests to be compared: originally proposed to target CVs or RVs (or both),

whether pooling over variants, whether sensitive to association directions (1/), to a large number of non-causal RVs

(nRVs) and to a few non-causal CVs (nCVs), requiring permutations for P-value calculations, capability to adjust for

other covariates (Cov), applicability to other non-binary traits, whether can be formulated as testing on a variance

component in a random-effects (R-E) model, and references for more details

Original Sens to Sens to Sens to Other

Test target Pool 1/ nRVs nCVs Permut Cov traits R-E Refs

Score CV No No No No No Yes Yes Yes 1

SSU CV No No No Yes No Yes Yes Yes 2

wSSU-P Both No No No No Yes Yes Yes Yes Here

SSUw CV No No No No No Yes Yes Yes 2

Sum CV No Yes Yes Yes No Yes Yes No 2

KMR CV No No No Yes No Yes Yes Yes 4, 5

CMC RV Yes Yes Yes No No No No No 6

wSum RV Yes Yes Yes Some Some No No No 7

aSum-P Both Yes Some Yes Some Yes Yes Yes No 8

Step-up RV Yes Some Some No Yes Yes Yes No 10

Seq-aSum Both Yes Some Some Yes Yes Yes Yes No Here

Seq-aSum-VS Both Yes Some Some No Yes Yes Yes No Here

KBAC RV No Some Some Some Yes Some No No 11

C-alpha-A RV No No No Yes No No No Yes 9

C-alpha-P RV No No No Yes Yes No No Yes 9

RBT RV Yes Some Yes No Yes No No No 12

Refs: 1, Clayton et al. [2004]; 2, Pan [2009]; 3, Conneely and Boehnke [2007]; 4, Kwee et al. [2008]; 5, Wu et al. [2010]; 6, Li and Leal [2008]; 7,

Madsen and Browning [2009]; 8, Han and Pan [2010]; 9, Neale et al. [2011]; 10, Hoffmann et al. [2010]; 11, Liu and Leal [2010]; 12, Ionita-Laza

et al. [2011].

association tests performed similarly and suffered from

substantial loss of power. Across all the situations, the

INDEPENDENT RVs SSU, KMR and C-alpha performed similarly and were

We first consider that there is no LD between any two RVs, most powerful. Although the three sequential model

mimicking the situation where mutations are all completely selection approaches (Step-up, Seq, Seq-VS), the KBAC

random and independent of each other. To investigate the and the aSum test performed well with no or few

possible dependence of performance on the significance level, non-causal RVs, surprisingly, as the number of non-causal

we used both a 5 0.05 and a 5 0.01. RVs increased, their performance deteriorated more

Table II shows that all the tests had satisfactory Type I than that of the SSU, KMR and C-alpha tests. Nevertheless,

error rates except that the C-alpha-A test might have the above procedures did improve over the pooled

some inflated Type I error rates at a 5 0.01 (as shown by association tests.

the bold values), suggesting that perhaps a larger sample It is noted that the CMC(0.01) test (with MAFr0.01 as

size is needed for using its asymptotic distribution with a the cut-off for RVs) was less powerful than the default

more stringent significance level. CMC, i.e. CMC(0.05), test in Table III because the former

For power comparison, the overall conclusions are the unnecessarily formed a few extra groups for CVs and

same with either a 5 0.05 and a 5 0.01. In each scenario three increased the DF of the test; in contrast, the former

most powerful ones are highlighted as bold values in each performed better than the latter in Table IV, presumably

table. First, for the non-null case that the eight causal RVs because some causal RVs might have an overall

shared a common OR (Table III), which is ideal for the MAF40.01 (due to their enrichment in cases) and the

pooled association tests (Sum, CMC, wSum), the pooled CMC(0.01) test grouped these causal RVs into separate

association tests and KBAC were most powerful if there groups, avoiding pooling them over with other causal RVs

were no or few non-causal RVs (i.e. RVs not associated with with opposite association directions and thus improving

the trait). As the number of non-causal RVs increased, the power. Hence, the choice of the MAF threshold for RVs is

SSU and KMR gradually became the most powerful while important for CMC, but it is unclear how to do so

the C-alpha test and the model selection approaches also generally in practice.

had much improved performance relatively. The KBAC was It is noted that since the true model was the main-effects

most powerful except for the case with 64 non-causal RVs. model (1), KMR with a linear kernel corresponded to using

Note that the aSum test maintained power as high as that of the true model, thus it was more powerful than using a

the Sum test, while the single SNV-based test, UminP, most quadratic kernel; the small performance difference between

commonly used in GWAS, had consistently low power. using the two kernels demonstrated the robustness of the

For the case that the association strengths of the KMR method. It is also noted that, since all the RVs were

causal RVs were not constant with possibly opposite independent, the covariance matrix V was nearly diagonal,

Genet. Epidemiol.

Statistical Tests for Rare Variants 613

TABLE II. Type I error rates at nominal level a based on 1,000 replicates for eight RVs plus a number of non-causal RVs

a 5 0.05 a 5 0.01

Test 0 4 8 16 32 0 4 8 16 32

U min P 0.027 0.027 0.016 0.011 0.019 0.003 0.001 0.004 0.001 0.002

Score 0.043 0.049 0.040 0.040 0.040 0.006 0.009 0.005 0.005 0.007

SSU 0.044 0.055 0.045 0.037 0.043 0.004 0.013 0.009 0.005 0.011

wSSU-P 0.052 0.051 0.048 0.048 0.046 0.008 0.008 0.014 0.010 0.008

SSUw 0.041 0.049 0.039 0.034 0.040 0.006 0.011 0.005 0.005 0.007

Sum 0.047 0.055 0.041 0.054 0.038 0.012 0.007 0.010 0.010 0.007

KMR (Linear) 0.046 0.056 0.046 0.042 0.047 0.007 0.016 0.011 0.007 0.012

KMR (Quad) 0.046 0.056 0.047 0.039 0.046 0.007 0.016 0.010 0.006 0.011

CMC (0.01) 0.035 0.053 0.044 0.055 0.039 0.008 0.014 0.010 0.011 0.009

CMC 0.048 0.053 0.043 0.056 0.051 0.010 0.009 0.011 0.011 0.007

wSum 0.050 0.057 0.038 0.059 0.056 0.010 0.012 0.011 0.009 0.006

aSum-P 0.058 0.064 0.052 0.063 0.047 0.012 0.011 0.010 0.010 0.011

Step-up 0.046 0.059 0.056 0.051 0.051 0.012 0.011 0.009 0.009 0.010

Seq-aSum 0.044 0.066 0.056 0.055 0.059 0.008 0.013 0.008 0.008 0.013

Seq-aSum-VS 0.050 0.058 0.056 0.051 0.058 0.011 0.018 0.011 0.009 0.013

KBAC 0.058 0.044 0.053 0.054 0.046 0.013 0.007 0.009 0.012 0.009

C-alpha-A 0.045 0.051 0.042 0.036 0.043 0.016 0.030 0.022 0.010 0.014

C-alpha-P 0.050 0.065 0.058 0.051 0.055 0.005 0.016 0.013 0.006 0.012

RBT 0.045 0.045 0.050 0.062 0.044 0.011 0.010 0.011 0.011 0.005

TABLE III. Empirical power for tests at nominal level a based on 1,000 replicates for an ideal case for eight causal RVs

with a common association strength OR 5 2 and a number of non-causal RVs

a 5 0.05 a 5 0.01

Test 0 4 8 16 32 64 0 4 8 16 32 64

U min P 0.441 0.336 0.296 0.222 0.175 0.117 0.142 0.089 0.094 0.050 0.043 0.029

Score 0.746 0.632 0.595 0.471 0.332 0.245 0.496 0.391 0.314 0.221 0.143 0.073

SSU 0.756 0.702 0.694 0.626 0.499 0.423 0.525 0.479 0.448 0.379 0.283 0.205

wSSU-P 0.821 0.732 0.714 0.644 0.514 0.390 0.573 0.471 0.407 0.332 0.222 0.161

SSUw 0.743 0.638 0.593 0.477 0.339 0.268 0.502 0.389 0.316 0.218 0.153 0.082

Sum 0.951 0.875 0.808 0.673 0.484 0.313 0.859 0.709 0.605 0.438 0.248 0.116

KMR (Linear) 0.762 0.711 0.699 0.631 0.509 0.438 0.548 0.500 0.473 0.405 0.308 0.234

KMR (Quad) 0.755 0.707 0.699 0.629 0.501 0.410 0.545 0.497 0.466 0.403 0.299 0.215

CMC (0.01) 0.853 0.761 0.702 0.628 0.484 0.396 0.672 0.524 0.452 0.384 0.268 0.218

CMC 0.938 0.853 0.777 0.616 0.399 0.211 0.831 0.679 0.570 0.383 0.196 0.086

wSum 0.940 0.846 0.782 0.618 0.424 0.267 0.838 0.687 0.568 0.394 0.216 0.114

aSum-P 0.933 0.858 0.780 0.669 0.499 0.313 0.781 0.611 0.534 0.381 0.257 0.125

Step-up 0.859 0.801 0.769 0.679 0.521 0.335 0.712 0.608 0.552 0.431 0.301 0.135

Seq-aSum 0.810 0.705 0.663 0.547 0.407 0.312 0.596 0.470 0.415 0.320 0.190 0.128

Seq-aSum-VS 0.798 0.722 0.692 0.590 0.420 0.344 0.598 0.506 0.452 0.345 0.216 0.141

KBAC 0.960 0.911 0.867 0.779 0.600 0.388 0.858 0.749 0.680 0.529 0.317 0.160

C-alpha-A 0.741 0.687 0.664 0.597 0.460 0.364 0.637 0.580 0.538 0.446 0.320 0.234

C-alpha-P 0.771 0.712 0.688 0.627 0.484 0.378 0.542 0.492 0.459 0.402 0.305 0.219

RBT 0.941 0.849 0.784 0.664 0.463 0.321 0.813 0.667 0.587 0.424 0.238 0.121

and thus the score test and SSUw test performed similarly. informative to weight the variants according to their

Finally, since in the current simulation set-up, causal RVs MAFs, suggesting why the SSU test outperformed the

were randomly chosen with various MAFs, it was not wSSU-P and SSUw tests.

Genet. Epidemiol.

614 Basu and Pan

TABLE IV. Empirical power for tests at nominal level a based on 1,000 replicates for a non-ideal case for eight causal

RVs with various association strengths OR 5 (3,3,2,2,2,1/2,1/2,1/2) and a number of non-causal RVs

a 5 0.05 a 5 0.01

Test 0 4 8 16 32 0 4 8 16 32

U min P 0.607 0.532 0.481 0.417 0.346 0.318 0.259 0.227 0.204 0.142

Score 0.869 0.772 0.721 0.632 0.483 0.660 0.532 0.480 0.356 0.233

SSU 0.895 0.835 0.815 0.774 0.696 0.723 0.662 0.645 0.583 0.472

wSSU-P 0.861 0.776 0.735 0.685 0.550 0.606 0.510 0.460 0.401 0.258

SSUw 0.867 0.773 0.732 0.633 0.501 0.661 0.550 0.481 0.355 0.238

Sum 0.682 0.566 0.465 0.365 0.258 0.471 0.348 0.257 0.172 0.101

KMR (Linear) 0.897 0.842 0.824 0.783 0.707 0.740 0.678 0.667 0.619 0.495

KMR (Quad) 0.893 0.835 0.815 0.781 0.698 0.734 0.680 0.663 0.608 0.484

CMC (0.01) 0.703 0.669 0.670 0.670 0.590 0.511 0.457 0.470 0.470 0.383

CMC 0.661 0.544 0.456 0.336 0.204 0.461 0.337 0.235 0.157 0.086

wSum 0.659 0.548 0.459 0.335 0.228 0.460 0.336 0.236 0.158 0.093

aSum-P 0.854 0.745 0.684 0.574 0.430 0.670 0.538 0.430 0.315 0.207

Step-up 0.839 0.767 0.724 0.640 0.527 0.652 0.564 0.518 0.413 0.285

Seq-aSum 0.892 0.811 0.757 0.671 0.528 0.752 0.620 0.532 0.438 0.273

Seq-aSum-VS 0.885 0.807 0.768 0.686 0.545 0.729 0.623 0.567 0.448 0.293

KBAC 0.907 0.813 0.763 0.642 0.436 0.737 0.607 0.536 0.399 0.199

C-alpha-A 0.892 0.826 0.802 0.757 0.655 0.824 0.732 0.720 0.653 0.512

C-alpha-P 0.906 0.844 0.823 0.775 0.674 0.735 0.673 0.661 0.612 0.496

RBT 0.810 0.659 0.603 0.482 0.301 0.590 0.429 0.356 0.250 0.125

TABLE V. Type I error (with OR 5 1) and power (with eight causal RVs with OR 5 (3,1/3,2,2,2,1/2,1/2,1/2)) for tests at

nominal level a 5 0.05 based on 1,000 replicates for eight RVs and a number of other non-causal RVs

OR 5 1 OR 5 (3,1/3,2,2,2,1/2,1/2,1/2)

Test 0 4 8 16 32 0 4 8 16 32

U min P 0.033 0.027 0.026 0.016 0.013 0.489 0.479 0.452 0.365 0.318

Score 0.034 0.022 0.025 0.019 0.023 0.599 0.538 0.491 0.380 0.276

SSU 0.040 0.041 0.052 0.044 0.036 0.603 0.624 0.635 0.581 0.574

wSSU-P 0.057 0.043 0.047 0.062 0.053 0.566 0.586 0.609 0.585 0.491

SSUw 0.035 0.042 0.049 0.033 0.034 0.532 0.561 0.574 0.506 0.493

Sum 0.049 0.047 0.059 0.033 0.049 0.342 0.312 0.315 0.258 0.239

KMR (Linear) 0.042 0.045 0.057 0.046 0.043 0.611 0.630 0.644 0.597 0.590

KMR (Quad) 0.038 0.033 0.041 0.030 0.025 0.545 0.563 0.565 0.493 0.474

CMC 0.045 0.053 0.056 0.036 0.060 0.296 0.283 0.189 0.182 0.365

wSum 0.045 0.054 0.056 0.040 0.063 0.369 0.297 0.287 0.191 0.200

aSum-P 0.050 0.046 0.061 0.038 0.053 0.350 0.323 0.325 0.258 0.243

Step-up 0.047 0.060 0.059 0.042 0.050 0.524 0.516 0.532 0.429 0.409

Seq-aSum 0.045 0.062 0.054 0.056 0.055 0.658 0.617 0.596 0.484 0.416

Seq-aSum-VS 0.043 0.056 0.058 0.054 0.049 0.658 0.606 0.577 0.472 0.414

KBAC 0.050 0.054 0.050 0.053 0.049 0.497 0.439 0.426 0.371 0.275

C-alpha-A 0.065 0.076 0.092 0.097 0.110 – – – – –

C-alpha-P 0.050 0.049 0.062 0.057 0.048 0.629 0.650 0.668 0.607 0.598

RBT 0.047 0.039 0.036 0.060 0.056 0.374 0.343 0.386 0.357 0.279

We next consider the case where all the RVs, both causal RVs since they were in LD. For the null case (Table V),

and non-causal ones, were possibly correlated. In this case, all the tests except C-alpha-A had their Type I error rates

Genet. Epidemiol.

Statistical Tests for Rare Variants 615

TABLE VI. Type I error (with OR 5 1) and power (with eight causal RVs with OR 5 (3,1/3,2,2,2,1/2,1/2,1/2)) for tests at

nominal level a 5 0.05 based on 1,000 replicates for eight RVs and a number of other non-causal RVs

OR 5 1 OR 5 (3,1/3,2,2,2,1/2,1/2,1/2)

Test 0 8 16 32 64 0 8 16 32 64

U min P 0.032 0.018 0.021 0.014 0.007 0.506 0.380 0.324 0.288 0.208

Score 0.029 0.029 0.028 0.019 0.021 0.631 0.480 0.373 0.241 0.160

SSU 0.049 0.051 0.035 0.034 0.034 0.642 0.553 0.475 0.444 0.334

wSSU-P 0.045 0.060 0.042 0.050 0.052 0.606 0.494 0.424 0.362 0.269

SSUw 0.045 0.040 0.027 0.015 0.036 0.562 0.450 0.352 0.272 0.187

Sum 0.046 0.059 0.046 0.046 0.046 0.345 0.229 0.159 0.110 0.079

KMR (Linear) 0.051 0.056 0.039 0.040 0.037 0.649 0.568 0.490 0.459 0.356

KMR (Quad) 0.046 0.049 0.022 0.021 0.017 0.572 0.487 0.392 0.331 0.205

CMC 0.046 0.053 0.040 0.050 0.047 0.339 0.235 0.193 0.124 0.111

wSum 0.048 0.052 0.041 0.053 0.048 0.342 0.237 0.199 0.133 0.114

aSum-P 0.052 0.061 0.049 0.046 0.052 0.364 0.239 0.170 0.113 0.081

Step-up 0.057 0.055 0.047 0.048 0.051 0.554 0.449 0.378 0.304 0.213

Seq-aSum 0.051 0.053 0.041 0.046 0.052 0.703 0.584 0.453 0.353 0.249

Seq-aSum-VS 0.053 0.053 0.048 0.041 0.054 0.701 0.572 0.447 0.351 0.258

KBAC 0.048 0.058 0.036 0.053 0.047 0.527 0.388 0.321 0.262 0.180

C-alpha-A 0.076 0.093 0.084 0.092 0.118 – – – – –

C-alpha-P 0.055 0.065 0.043 0.050 0.047 0.669 0.585 0.504 0.472 0.340

RBT 0.057 0.059 0.049 0.042 0.054 0.376 0.285 0.188 0.141 0.097

There is LD among the 8 RVs and among other non-causal RVs, but no LD between the 8 RVs and non-causal RVs.

well controlled. Since the asymptotic distribution of the INDEPENDENT RVs AND CVs

C-alpha test is derived under the assumption that all the RVs Finally we considered the case with independent RVs

are independent, which was violated here, one has to use its and four non-causal CVs (with MAFs randomly between

permutational distribution, which appears to work well. 0.01 and 0.1). Although the aSum test was proposed by

For the non-null case with varying association strengths Han and Pan [2010a] to group CVs separately from RVs, as

(Table V), again all the pooled tests suffered from done in CMC, for simplicity here, we did not do such

significant power loss, while the SSU, KMR and C-alpha-P groupings. All the tests had satisfactory Type I error rates

tests were most powerful. The three sequential model (not shown). For power comparison (Table VII), it is most

selection approaches and KBAC performed similarly and notable that the SSU, KMR and C-alpha tests were all low-

better than the aSum test, and all improved over the powered, due to the undue influence of the CVs, as

pooled association tests. analyzed before. The performance of the pooled associa-

Due to the LD among the RVs, the score test and SSUw tion tests and KBAC also degraded. With CVs, variable

test performed differently: When there was no non-causal selection worked well as evidenced by the good perfor-

RVs, the score test was more powerful; however, as the mance of the Step-up procedure, and by that the Seq-

number of non-causal RVs increased, the SSUw test aSum-VS performed much better than Seq-aSum without

became much more powerful than the score test. variable selection. We also note that the weighted C-alpha

tests were much more powerful than the original

unweighted C-alpha test; between the two weighted

C-alpha tests, the first one with weights inversely

NO LD BETWEEN CAUSAL RVs AND NON- proportional to the MAFs performed much better than

CAUSAL RVs the second one in the presence of a large number of non-

Now we consider the case where causal RVs were causal RVs, but not so otherwise; neither was more

correlated, non-causal RVs were also correlated, but there powerful than the wSSU-P test. Overall, the weighted

was no LD between causal and non-causal RVs. For the SSU test (wSSU-P) performed best, closely followed by the

null case (Table VI), again all the tests except the C-alpha- Step-up procedure, then by the SSUw and score tests.

A had satisfactory Type I error rates. The C-alpha-A did

not work because its independence assumption on the RVs

was violated.

For power comparison, again due to the presence of DISCUSSION

opposite association directions, the pooled association

tests performed similarly and had the lowest power. With The three pooled association tests (i.e. Sum, CMC and

no or few non-causal RVs, Seq-aSum and Seq-aSum-VS wSum) performed similarly for RVs. They were most

performed best, closely followed by the C-alpha-P, KMR powerful when there were no opposite association direc-

and SSU tests; for a larger number of non-causal RVs, the tions and when there were no or only few non-causal RVs;

C-alpha-P, KMR and SSU tests were the winners. otherwise, they suffered from a substantial loss of power.

Genet. Epidemiol.

616 Basu and Pan

TABLE VII. Empirical power for the tests at nominal level a 5 0.05 based on 1,000 replicates with eight causal RVs, four

neutral CVs and a number of other neutral RVs

OR 5 (2,2,2,2,2,2,2,2) OR 5 (3,3,2,2,2,,1/2,1/2,1/2)

Test 0 4 8 16 32 0 4 8 16 32

U min P 0.355 0.283 0.269 0.213 0.156 0.518 0.482 0.441 0.412 0.331

Score 0.628 0.580 0.498 0.424 0.348 0.766 0.706 0.629 0.584 0.466

SSU 0.148 0.128 0.134 0.131 0.135 0.225 0.201 0.206 0.203 0.215

wSSU-P 0.777 0.729 0.700 0.589 0.518 0.810 0.764 0.724 0.655 0.582

SSUw 0.634 0.592 0.515 0.429 0.332 0.765 0.704 0.631 0.599 0.489

Sum 0.455 0.438 0.396 0.348 0.299 0.231 0.225 0.195 0.199 0.152

KMR (Linear) 0.158 0.138 0.151 0.145 0.153 0.237 0.216 0.222 0.223 0.234

KMR (Quad) 0.153 0.124 0.136 0.137 0.141 0.219 0.198 0.204 0.201 0.219

CMC 0.575 0.512 0.429 0.309 0.212 0.296 0.254 0.209 0.155 0.124

wSum 0.533 0.508 0.469 0.408 0.346 0.291 0.285 0.249 0.230 0.181

aSum-P 0.467 0.457 0.414 0.355 0.310 0.239 0.245 0.206 0.202 0.158

Step-up 0.776 0.750 0.715 0.610 0.522 0.727 0.712 0.658 0.605 0.499

Seq-aSum 0.368 0.314 0.323 0.300 0.266 0.453 0.410 0.392 0.395 0.342

Seq-aSum-VS 0.550 0.518 0.502 0.450 0.379 0.610 0.617 0.567 0.541 0.471

KBAC 0.554 0.537 0.478 0.446 0.370 0.415 0.402 0.358 0.335 0.270

C-alpha-A 0.106 0.083 0.089 0.088 0.082 0.165 0.154 0.146 0.149 0.160

C-alpha-P 0.165 0.150 0.145 0.139 0.139 0.245 0.233 0.228 0.220 0.225

w1C-alpha-P 0.542 0.527 0.527 0.496 0.474 0.670 0.642 0.632 0.636 0.593

w2C-alpha-P 0.628 0.568 0.476 0.388 0.298 0.773 0.698 0.606 0.563 0.422

RBT 0.826 0.770 0.688 0.592 0.453 0.630 0.581 0.487 0.410 0.321

Perhaps the most surprising and interesting finding is harmful. As shown by the close performance between

that, overall, in the presence of opposite association Seq-aSum and Seq-aSum-VS, there is only minimal gain or

directions and non-causal RVs, the SSU, KMR and loss in selecting causal RVs. On the other hand, as shown

C-alpha-P performed similarly and best. Although the here, when there were both protective and deleterious

three methods appear quite different, they share a causal RVs and few non-causal RVs, our newly proposed

common feature: all can be regarded as testing on a Seq-aSum and Seq-aSum-VS were or nearly were the most

variance component in a R-E model, thus are robust to a powerful, suggesting their applicability not only to RVs,

large number of parameters induced by a large group of but also to CVs: in analyzing multiple common SNVs in an

RVs. This is related to the success story of the class of gene LD region, if the untyped causal SNV is in LD with the

set tests, including both the SSU-equivalent Goeman’s test multiple typed SNVs, the two methods could be powerful.

[Goeman et al., 2004] and KMR [Liu et al., 2008], applied to In addition, leveraging on the idea of pooling and thus

high-dimensional microarray data. Furthermore, the SSU reduced degrees of freedom, they can be also applied to

test and KMR are themselves closely related to each other detect epistasis, as done in He et al. [2010].

[Pan, 2011], and share some advantages: they can be Several approaches are not considered here, including

applied to other GLMs for other types of traits, such as penalized regression [e.g. Malo et al., 2008 for CVs, Zhou

quantitative or survival traits, and to adjust for other et al., 2010 for both CVs and RVs] and some nonparametric

covariates, such as environmental factors, which are regression techniques, such as logic regression [Kooperberg

important as argued by Bansal et al. [2010]. et al., 2001] for CVs, and a Bayesian GLM [Yi and Zhi,

The approaches based on model selection (aSum, 2011], largely due to their difficulty in controlling Type I

Step-up, Seq-aSum, Seq-aSum-VS) improve over the error rates (which is required to make a formal and fair

pooled association tests in the presence of opposite comparison with other statistical tests) and/or associated

association directions. However, in spite of their strong high computing cost, e.g. in permutation tests, especially if

motivation for model selection, their performance might one aims to take account of the uncertainty in choosing

not be as impressive as expected, especially in the presence optimal penalization or tuning parameters. Penalized

of a large number of non-causal RVs. A possible explana- regression and logic regression belong to the class of

tion lies in the trade-off between the gain and the cost of model selection-based approaches. Compared to the four

model selection: in spite of possibly a strong association selection methods compared here, penalized regression

with the trait, due to its low frequency of any single RV, and logic regression are believed to have some advantages.

often there is only minimal power to detect its own However, existing penalized regression and logic regres-

association with the trait, rendering it difficult to sion methods do not incorporate the strategy of collapsing

distinguish whether the RV is or is not associated with RVs or of R-E models, two key elements for the success of

the trait, and if so, whether its effect is protective or the compared methods for RVs; further studies are needed

Genet. Epidemiol.

Statistical Tests for Rare Variants 617

to evaluate their performance. Another approach is based environmental factors and to detect environment-gene

on haplotype inference [Zhu et al., 2010; Li et al., 2010], interactions, their applicability to CVs and/or RVs no

which is appealing for its applicability to GWAS for matter whether they are in LD or not. We note that the SSU

association analysis of more frequent RVs in the MAF test can be applied to more complex regression models,

range of 0.1–5%. e.g. with both main effects and some interaction terms;

We note that Price et al. [2010] proposed using multiple equally, in KMR we can use a kernel that can capture some

thresholds and (possibly predicted) biological functional complex interactions among the SNVs. Of course, as

annotations to group RVs and empirically showed its shown earlier, with any given kernel and its decomposi-

advantage over using only one group. For simplicity, we tion, we can have an SSU test equivalent to KMR.

have only considered the use of a single group. However, It would be of interest to compare the performance of

our conclusion should be useful for the case with multiple the SSU/KMR and KBAC in the presence of interactions

groups for RVs: a test with high power for a single group is among RVs.

likely to be even more powerful for multiple groups that A potentially useful resource resulting from this work is

are appropriately constructed, as shown by Pan and Shen freely available software: we have implemented most of the

[2011]. As shown by our simulation studies, mixing non- compared methods in R; R code will be posted on our web

causal CVs (or RVs with relatively higher MAFs) with RVs site at http://www.biostat.umn.edu/weip/prog.html.

may degrade the performance of several tests, especially

the SSU, KMR, C-alpha and KBAC tests. Hence it is a ACKNOWLEDGMENTS

critical question in practice how to define RVs, to which a

test is applied. There are two possible ways. The first way

W.P. thanks Drs. Roeder and Witte for sharing their

is to use the multiple cut-offs of MAF to define RVs and

manuscripts, Drs. Wu, Ionita-Laza and Leal and Gao Wang

then combine the test results, as implemented in the

for sharing their computer programs. The authors are

multiple threshold test of Price et al. [2010] and in the

grateful to the reviewers for helpful and constructive

adaptive tests of Pan and Shen [2011]. Second, as shown

comments. This research was partially supported by

here and by other authors [Madsen and Browning, 2009],

NIH grant R21DK089351; W.P. was also supported by

weighting the variants in a test with suitably chosen

R01HL65462 and R01HL105397.

weights (e.g. inversely proportional to their MAFs) may

improve the performance of the test. We have not

investigated these issue extensively and more studies are

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APPENDIX: RELATIONSHIPS

Morgenthaler S, Thilly WG. 2007. A strategy to discover BETWEEN GOEMAN’S TEST AND

genes that carry multi-allelic or mono-allelic risk for

common diseases: a cohort allelic sums test (CAST). Mutat Res ZELTERMAN AND CHEN’S

615:28–56.

Morris AP, Zeggini E. 2010. An evaluation of statistical approaches to

HOMOGENEITY TEST

rare variant analysis in genetic association studies. Genet Epide-

miol 34:188–193. We first review Zelterman and Chen’s homogeneity test.

Neale BM, Rivas MA, Voight BF, Altshuler D, Devlin B, Ogho- Suppose that y1 ; . . . ; yn are independent random variables

Melander M, Katherisan S, Purcell SM, Roeder K, Daly MJ. 2011. with respective pdf’s fi ðyi jli Þ, conditional on a k-dimen-

Testing for an unusual distribution of rare variants. PLoS Genet sional parameter lt. Under H0, all li’s are equal to a fixed

7:e1001322. vector l0. It is assumed that li’s are random: li ¼ l0 1az,

Neyman J, Scott E. 1966. On the use of c-alpha optimal tests of where z is a k-dimensional random variable with E(z) 5 0

composite hypothesis. Bull Int Stat Inst 41:477–497. and CovðzÞ ¼ ¼ ðsst Þ. Under this formulation, testing H0

Genet. Epidemiol.

Statistical Tests for Rare Variants 619

0 0

is equivalent to testing H00 : a 5 0. Zelterman and Chen Since EðUðiÞ UðjÞ Þ ¼ EðUðiÞ ÞEðUðjÞ Þ ¼ 0, by ignoring

[1988] showed that the score test statistic for H00 is cross-product terms UðiÞ UðjÞ , we have Goeman’s test

statistic

1X n X k

@2 fi ðyi jl0 Þ 1

TZ ¼ sss 1 1 1X n X

2 i¼1 s¼1 @l20s fi ðyi jl0 Þ S ¼ U 0 U trðVÞ ¼ 00

sst fi;st =fi ¼ TZ :

2 2 2 i¼1 s;t

X

n X

@2 fi ðyi jl0 Þ 1

1 sst ;

i¼1 sot

@l0s @l0t fi ðyi jl0 Þ Hence, Goeman’s test is equivalent to Zelterman and

Chen’s homogeneity test, which covers the C-alpha test as

where l0 ¼ ðl01 ; . . . ; l0k Þ0 .

a special case (with mi as yi and f i as Bin(ni,pi)). By the

For observation Yi, the score vector is UðiÞ ¼

equivalence among permutation-based Goeman’s test,

0

ðfi;1 0

=fi ; . . . ; fi;k =fi Þ0 , and

X SSU test and KMR test with a linear kernel, we know that

0

UðiÞ 0

UðiÞ ¼ 0 0

sst fi;s fi;t : the SSU test, KMR test with a linear kernel, and

s;t permutation-based C-alpha test are all equivalent (if a

0 common random variable of interest is modeled). How-

For simplicity we use notation fi;s ¼ @fi =@l0s. On the other ever, in the current context, since the disease status of

hand, we have the (s,t) th element of V as subject i is treated as random variable of interest yi in the

X

n

@UðiÞ;s X

n 00

fi;st 0 0

fi fi;s fi;t SSU test and KMR, while the mutation status of variant i is

Vst ¼ ¼ : treated as yi in the C-alpha test, the three tests are closely

I¼1

@l0t I¼1

fi2 related but not exactly equivalent.

Genet. Epidemiol.

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