Zhang et al.

BMC Bioinformatics (2023) 24:2 BMC Bioinformatics

https://doi.org/10.1186/s12859-022-05114-x

RESEARCH Open Access

A gene based combination test using GWAS

summary data
Jianjun Zhang1, Xiaoyu Liang2, Samantha Gonzales1, Jianguo Liu1, Xiaoyi Raymond Gao3 and Xuexia Wang4*

*Correspondence:
[email protected] Abstract
1
Department of Mathematics, Background: Gene-based association tests provide a useful alternative and comple-
University of North Texas, 225 ment to the usual single marker association tests, especially in genome-wide associa-
Avenue E, Denton, TX 76201, USA tion studies (GWAS). The way of weighting for variants in a gene plays an important
2
Department of Epidemiology
and Biostatistics, Michigan State role in boosting the power of a gene-based association test. Appropriate weights can
University, 909 Wilson Rd Room boost statistical power, especially when detecting genetic variants with weak effects
B601, East Lansing, MI 48824, on a trait. One major limitation of existing gene-based association tests lies in using
USA
3
Department of Ophthalmology weights that are predetermined biologically or empirically. This limitation often attenu-
and Visual Science, Department ates the power of a test. On another hand, effect sizes or directions of causal genetic
of Biomedical informatics, variants in real data are usually unknown, driving a need for a flexible yet robust meth-
Division of Human Genetics,
Ohio State University, 915 odology of gene based association tests. Furthermore, access to individual-level data is
Olentangy River Road, Columbus, often limited, while thousands of GWAS summary data are publicly and freely available.
OH 43212, USA
4
Department of Biostatistics, Results: To resolve these limitations, we propose a combination test named as OWC
Robert Stempel College of Public which is based on summary statistics from GWAS data. Several traditional methods
Health and Social Work, Florida including burden test, weighted sum of squared score test [SSU], weighted sum statis-
International University, 11200
SW 8th street, Miami, FL 33174, tic [WSS], SNP-set Kernel Association Test [SKAT], and the score test are special cases of
USA OWC. To evaluate the performance of OWC, we perform extensive simulation stud-
ies. Results of simulation studies demonstrate that OWC outperforms several existing
popular methods. We further show that OWC outperforms comparison methods in
real-world data analyses using schizophrenia GWAS summary data and a fasting glu-
cose GWAS meta-analysis data. The proposed method is implemented in an R package
available at https://​github.​com/​Xuexia-​Wang/​OWC-R-​packa​ge
Conclusions: We propose a novel gene-based association test that incorporates four
different weighting schemes (two constant weights and two weights proportional to
normal statistic Z) and includes several popular methods as its special cases. Results
of the simulation studies and real data analyses illustrate that the proposed test, OWC,
outperforms comparable methods in most scenarios. These results demonstrate that
OWC is a useful tool that adapts to the underlying biological model for a disease by
weighting appropriately genetic variants and combination of well-known gene-based
tests.
Keywords: Combination test, Score test, Burden test, Weighted sum of squared score
test, Weighted sum statistic

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Zhang et al. BMC Bioinformatics (2023) 24:2 Page 2 of 19

Background
To date, genome-wide association studies (GWAS) have identified more than thousands
of genetic variants associated with complex traits or diseases. However, these identified
genetic variants only can explain a small to modest fraction of heritability [1]. To identify
genetic variants which can explain the missing heritability, people need to use data with
larger sample size and/or more powerful statistical tests, especially when causal genetic
variants have weak effects on complex traits. In reality, it is often difficult to access
patients data directly, and thus difficult to obtain data with sufficiently large sample size.
On the other hand, thousands of GWAS summary data are publicly and freely available.
These GWAS data including p-values, effect sizes, directions of effects, or estimated sta-
tistics for single nucleotide polymorphisms (SNPs) motivate us to develop novel power-
ful methods for further analysis of GWAS summary data. The gene-based association
test using GWAS summary statistics can be viewed as a complementary approach to the
traditional single marker association test in GWAS.
When testing for genetic associations with a gene-based test, proper weights can
boost power substantially. However, one major limitation of existing gene-based asso-
ciation tests lies in using weights predetermined biologically or empirically. This limita-
tion often attenuates the power of a test. For example, both the burden test [2, 3] and
the weighted sum of squared score (SSU) test [4] are typical combination methods. The
burden test sets the same weight for each genetic variant, while the SSU test uses the
Z-score as a weight for each genetic variant. The presence of non-associated SNPs in a
gene can diminish the power of a test dramatically if an effective SNP selection method
or weighting method is not adopted [5]. The SSU method is robust and powerful when
there are protective, risk, and null variants in a considered region, but it is less powerful
than the burden test when a large number of genetic variants in the considered region
are causal and the direction of effects are the same. A statistical challenge is that the true
association patterns are usually unknown. A test may perform well for one real dataset,
but it may be less powerful for another dataset. There is no uniformly most powerful test
which is powerful in every situation [6]. In this study, we intend to develop a test which
is more powerful than well-known existing methods in most situations.
The power of a gene-based test depends on the underlying genetic architecture of a
complex trait. For different traits, the genetic architecture can differ in number, loca-
tion, effect size, and direction of effect for causal genetic variants in different genes. To
circumvent the difficulties in gene-based association test, we propose the combination
method, which is a general, flexible, and powerful method. When testing for weak asso-
ciations caused by small effect sizes or low frequency common genetic variants, the pro-
posed method performs significantly better than several popular gene based tests such
as sum test (ST) [7], squared sum test (S2T) [7], adaptive test (AT) [7], adaptive sum of
powered score tests (aSPU) [6], Gene-based Association Test using extended Simes pro-
cedure (GATES) [8] and sumSTAAR method which provides a framework for combin-
ing a wide range of gene-based association tests using summary statistics [9].
Testing association between a phenotype and a gene based on individual level data
(i.e. genotypes) of genetic variants in the gene is the same as testing association between
the phenotype and the gene based on summary statistics (i.e. Z-scores) in that gene [10,
11]. In the Methods section, we illustrated this conclusion with a score test framework.
 Zhang et al. BMC Bioinformatics

Table 1 Summary of the proposed score test Ss and its special cases
Method Description Weight Test Statistic
(2023) 24:2

′ ′
General method Score Test Ss Maximum of the weighted W = Z R−1 Ss = Z R−1 Z
sum of Z scores
′
Special cases of the Special case 1: Sum of Weighted sum of Z scores, W = R−1 Z, R = I SQ = Z Z
score test Ss Squared Score Statistic weights are Z scores
(SSU)
′
Special case 2: SNP-set Weighted sum of Z scores, W = R−1 Z, R = diag(a1 , . . . , am ), am ∼ beta(1, 25) SSKAT = Z R−1 Z
(Sequence) Kernel Asso- weights are weighted Z
ciation Test (SKAT) scores
′
Special case 3: PathSPU(2) Weighted sum of Z scores, W = R−1 Z, R = diag(a1 , . . . , am ), am are gene derived SpathSPU(2) = Z R−1 Z
weights are eQTL weighted weights
Z scores
Special case 4: Sum of Weighted sum of Z scores, M γ
m=1
W = Z γ −1 SPU(γ ) = Z m , γ = 1, 2, . . . , 8, ∞
Powered Score (SPU): weights are function of Z
Data-adaptive weighted scores
combination test.
′
Special case 5: Burden test Weighted sum of Z scores, Zm
M
W = (1, . . . , 1) LB = L(1, . . . , 1) = m=1
weights are all 1s
Special case 6: Weighted Weighted sum of Z scores, 1 1 ′ 1 1 1
,..., ,..., )
M
√ ) , where pm is the √
m=1
√
1 1) pm (1−pm ) 1 (1−p1 ) pm (1−pm ) pm (1−pm )
W = ( √p (1−p LW = L( √p = · Zm
Sum Statistic weights are related to MAFs MAF
Page 3 of 19
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 4 of 19

Furthermore, we proposed a new score test Ss which can reach its maximum when
′
weights of genetic variants is Z R−1 where Z is the Z score summary statistics and R
is the correlation matrix of genetic variants. Six existing methods can be considered as
its special cases and are summarized in Table 1. As indicated in Table 1, six gene-based
association tests based on individual level data can be easily modified to gene-based
association tests based on GWAS summary data [11]. Based on the score test and other
three typical methods, we propose a novel and powerful gene-based association test
using GWAS summary data, named as OWC, which can reaching its maximum through
finding the appropriate weights for the combination of the four tests. The burden test,
SSU, weighted sum statistic (WSS) [13], and score test are special cases of the proposed
OWC method. Furthermore, we show that OWC is more powerful than other compar-
ison methods in most simulation studies and identifies more trait associated genes in
three real datasets.
To evaluate the performance of the proposed method, we have conducted extensive
simulation studies and real data analyses. We compared our method, OWC, with six
existing comparable methods: (1) sum test (ST) [7]; (2) squared sum test (S2T) [7]; (3)
adaptive test (AT) [7]; (4) adaptive sum of powered score tests (aSPU) [6]; (5) Gene-
based Association Test using extended Simes procedure (GATES) [8]; and (6) sum-
STAAR [9]. All of the comparison methods are designed for testing associated genes for
a single trait. ST can be considered as a burden test statistic [12], S2T can be considered
as a quadratic test similar as the SNP-set kernel association test (SKAT) [13], and AT is a
combination of burden and quadratic tests, which is equivalent to the SKAT-O test [14].
The aSPU method chooses the most powerful test from a group of tests. GATES adopts
an extended Simes procedure and uses GWAS summary statistics to correct for multiple
testing issues and estimate the p-value promptly. sumSTAAR creates a frameworks to
combine multiple gene based tests with ACAT method [15].
Our proposed method OWC is more powerful than the six comparable tests in most
of the simulation scenarios. We further applied OWC and the other six tests to real data-
sets: (1) the GWAS summary data of schizophrenia (SCZ), which was obtained from the
Psychiatric Genomics Consortium (PGC); (2) the GWAS meta-analysis summary data
for fasting glucose, obtained from the European DIAMANTE study (a component of the
UK Biobank). The results of the real data analyses demonstrate that OWC is the most
effective test as it identified more trait-associated genes than other methods.

Results
Comparison of methods
The performance of the proposed method OWC are compared with six existing gene-
based association tests: the sum test (ST), the squared sum test (S2T), adaptive test (AT)
proposed by Guo and Wu [7], the adaptive sum of powered score tests (aSPU) method
proposed by Kwak and Pan [6], the Gene-based Association Test that uses Extended
Simes procedure (GATES) proposed by Li et al. [8], and the sumSTAAR [9].
Consider a gene with M genetic variants. Assume GWAS summary statistics such as Z
scores are available for all the genetic variants in the gene. Denote Zm , m = 1, 2, . . . , M
as the Z score of the mth variant. The six methods for testing genetic association are
described briefly as follows:
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 5 of 19


1 Sum test (ST), B = M m=1 Zm, which is similar as the burden test [12].

2 Squared sum test (S2T), Q = M m=1 Zm, which is a special case of the SKAT method
2

[13]. The squared sum test (S2T) is equivalent to the weighted sum of squared score
(SSU) statistic [4].
3 Adaptive test (AT), T = minρ∈[0,1] P(Qρ ), where Qρ = (1 − ρ)Q + ρB2, P(Qρ )
denotes the corresponding p-value.
4 Adaptive sum of powered score tests (aSPU), aSPUs= minγ ∈Ŵ PSPUs(γ ), where
 γ
SPUs(γ ) = M m=1 Zm,where γ is an integer.
5 Gene-based association test that uses extended Simes procedure (GATES),
m p 
pGATES = min mee(j)(j) , where p(j) is the j th smallest p-value, me(j) is the effective
number of independent p-values among the top j SNPs, me is the effective number of
independent p-values among the total M SNPs.
6 sumSTAAR combines p values of burden test, SKAT, SKAT-O [14], aggregated
Cauchy association test (ACAT-V) [15], the tests using functional linear regression
model (FLM) and principal component analysis (PCA) with ACAT method [15].

Denote Z ∼ MVN(0, R) where R is the linkage disequilibrium (LD) matrix of the gene,
′ ′
B = 1M Z ∼ N (0, 1M R1M ), where 1M denotes a column vector of length M with ele-
B2 ′
ments 1s. ′ follows χ1 distribution. The squared sum test Q=Z Z asymptotically
1M R1M
follows χ 2 distribution which is equivalent to the weighted sum of independent χ1 dis-
tributed random variables where the weights are the eigenvalues of R . The p-value of the
adaptive test T can be efficiently and simply computed by employing a one-dimensional
numerically search over ρ ∈ (0, 0.01, 0.04, 0.09, 0.16, 0.25, 0.5, 1) following Wu et al. [16].
The three test ST, S2T, and AT can be obtained using the “sats” function in the “mkatr”
package in R. Monte Carlo simulations are used to obtain the p-value of aSPU which can
be obtained using the “aSPUs” function in the “aSPU” R package. The GATES method
can be obtained from “gates” function in the “COMBAT” R package. sumSTAAR can be
obtained from the sumFREGAT package (function sumSTAAR() in sumFREGAT
v.1.2.3). When using the sumSTAAR method, we set the tests argument as the default
tests - burden test, SKAT, and ACAT.

Simulation studies
We conducted extensive simulation studies to evaluate the performance of the proposed
method OWC. Following the simulation settings in Guo and Wu [17], we performed the
type I error and power comparisons between OWC and the six comparable methods.
Estimating LD among genetic variants using any reference data from the same ancestry
is mostly accurate with an estimated inflation factor close to 1 [6]. Because of this, we
estimated the LD between genetic variants in a gene using the haplotypes with ances-
try from northern and western Europe (CEU) obtained from the 1000 Genomes project
[18].

Type I error
To evaluate the type I error, we obtain similar Z scores as in GWAS summary data from
a multivariate normal distribution MVN(0, R), where R denotes the corresponding LD
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 6 of 19

Table 2 Ratio of estimated type I error rates by the significance level for different test methods
α-level ST S2T AT GATES aSPU sumSTAAR​ OWC

1 × ­10−3 1.02 1.02 1.02 1.04 1.03 0.86 1.01

1 × ­10−4 1.00 1.03 1.00 1.03 1.02 1.10 1.04
1 × ­10−5 1.00 1.04 0.99 1.05 1.08 2.50 1.00
2.5 × ­10−6 1.04 1.14 1.00 0.92 1.04 3.00 1.08
2.8 × ­10−6 1.10 1.07 0.97 1.00 1.01 3.57 1.02
Notes: The comparison methods sum test(ST), squared sum test (S2T) and adaptive test (AT) mentioned in the paper
are equivalent to the methods burden test (LB ), the sum of squared score test (SQ ) and the combination of LB and SQ ,
respectively. Let SS denote the score test and LW denote the weighted sum statistic. The proposed combination method
OWC is a combination of LB , LW , SS , and SQ . sumSTAAR is a fexible framework for gene-based association studies using
GWAS summary statistics

matrix of gene EPB41. Gene EPB41 colocalizes with AMPA receptors which is thought
to interact with the cytoskeleton [19]. Abnormalities of brain-region in the expression
of subunits of the AMPA subtype of glutamate receptors in Schizophrenia patients have
been identified [20]. As in real data analysis, we first remove rare variants on gene EPB41
from our analysis and keep 11 SNPs with minor allele frequency (MAF) in the range
from 0.067 to 0.453 in the simulation studies. The LD matrix R of gene EPB41 is esti-
mated by using the 1000 Genomes Project reference panel [18]. Additional file 1: Link-
age disequilibrium matrix of gene EPB41. Fig. S1 shows the LD matrix with pairwise
correlations for the 11 SNPs in EPB41. Coefficients of five pairwise LD (r 2) are greater
than 0.5, and the others’ are less than 0.5. We use [21] to simulate the effect size beta
of a causal genetic variant and its standard error for the sumSTAAR method. To mimic
the real schizophrenia data used in Real Data Analysis section, we use the numbers of
cases as 13,833 and the number of controls as 18,310 as inputs to the simulated_vbeta
function and adjust “gamma.W” based on various simulation scenarios. We evaluated
the proposed method by using five different significance levels: α = 10−3 , 10−4 , 10−5,
2.5 × 10−6 and 2.80 × 10−6. In the simulations, p-values of the proposed method and
aSPU are estimated with 1 ­ 07 times replications. The type I error rates are estimated
7
based on 1­ 0 replications. Table 2 shows that the type I error rates of all of the methods
are well controlled except that there is slight type I inflation of the sumSTAAR method.

Power analysis
We further conduct extensive simulations to evaluate the power of the proposed
method. We consider different scenarios in terms of different number of causal
genetic variants, effect sizes and directions of causal variants, different number of
SNPs, LD structure, and allele frequency spectrum of the considered region. Gene
EPB41 contains 11 common SNPs. The range of the minor allele frequencies is (0.067,
0.453). The coefficients of five pairwise LD ( r 2 ) are greater than 0.5 in EPB41 (Addi-
tional file 1: Linkage disequilibrium matrix of gene EPB41. Fig. S1). We simulate ­104
summary statistics from MVN(A × △, R) where A denotes the directions of effects of
causal variants (i.e. risk or protective effect), △ denotes different settings of the effect
sizes of causal variants. R denotes the corresponding LD matrix of EPB41. We ran-
domly select a number of SNPs (e.g. 2, 3, 4, or 5) as causal variants from EPB41. For
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 7 of 19

Table 3 Power comparison between OWC and the other six tests. Data are simulated from
N(A × △, R). A has three or four nonzero elements with different signs which represent whether
the causal variants are risk or protective. △ denotes the different settings of effect sizes. R is the
corresponding LD matrix of gene EPB41. Power (%) is estimated under 2.5 × 10−6 significance level
No. causal nonzero △ nonzero A AT S2T ST GATES aSPU sumSTAAR​ OWC
variants

3 U(1,5) (1,1,1) 85.0 35.0 86.0 55.2 85.5 54 96.5

3 U(2,6) (1,1,− 1) 71.0 70.5 17.0 58.0 67.0 50.5 85.5
3 U(2,6) (1,− 1,− 1) 70.0 68.5 17.0 65.5 63.5 52.3 87.5
3 N(3,4) (1,1,1) 82.5 60.0 83.0 74.0 87.0 71.8 94.0
3 N(3,4) (1,1,− 1) 68.0 69.0 32.5 72.0 69.0 68.6 82.0
3 N(3,4) (1,− 1,− 1) 65.5 60.5 32.0 76.0 73.0 67.8 86.5
3 (4,2,1) (1,1,1) 64.0 3.5 68.5 13.5 78.5 7.9 95.0
3 (4,2,1) (1,1,− 1) 18.0 3.0 18.5 12.5 31.5 16.1 79.0
3 (4,2,1) (1,− 1,− 1) 3.0 4.0 0.5 12.0 8.5 9.0 76.0
3 (8,4,2) (1,1,1) 99.0 98.0 99.5 98.5 98.5 96.5 99.5
3 (8,4,2) (1,1,− 1) 97.0 96.5 98.5 94.5 98.5 93.9 99.0
3 (8,4,2) (1,− 1,− 1) 96.0 96.0 0.5 94.0 97.0 87.5 98.0
3 (4,4,2) (1,1,− 1) 69.0 45.0 45.0 28.5 65.6 43.5 93.5
3 (2,5,4) (1,− 1,− 1) 92.5 74.5 76.0 65.5 62.0 79.3 99.5
4 (4,4,2,1) (1,1,1,1) 97.0 47.5 98.5 24.5 98.5 53.2 99.5
4 (4,4,2,1) (1,1,1,− 1) 95.5 47.0 95.5 24.5 97.5 50.9 99.0
4 (4,4,2,1) (1,1,− 1,− 1) 71.0 45.0 0.5 24.0 70.5 52.2 93.0
5 (4,4,4,2,1) (1,1,1,1,1) 100.0 84.5 99.5 35.5 99.5 87.2 100.0
5 (4,4,4,2,1) (1,1,1,1,− 1) 99.5 84.0 99.0 35.0 98.5 82.1 99.5
5 (4,4,4,2,1) (1,1,1,− 1,− 1) 98.0 84.0 99.0 34.0 98.5 81.7 99.5
The comparison methods sum test(ST), squared sum test (S2T) and adaptive test (AT) mentioned in the paper are equivalent
to the methods burden test (LB ), the sum of squared score test (SQ ) and the combination of LB and SQ , respectively. Let SS
denote the score test and LW denote the weighted sum statistic. The proposed combination method OWC is a combination
of LB , LW , SS , and SQ . sumSTAAR is a fexible framework for gene-based association studies using GWAS summary statistics

a given gene, we randomly set the effects of the causal variants by drawing the cor-
responding number of elements of A equal to 1 or − 1, and set the effects of other
variants as 0. Table 3 shows the estimated power under three combinations of A for
different settings of △: a set of fixed values of △, two randomly simulated △ where
one is from uniform distribution, and the other from normal distribution. We use
2.50 × 10−6 as the significance level to claim a significant finding.
Table 3 shows that the proposed method OWC performs robustly well across all sce-
narios. It has the highest power in almost all of the scenarios when compared to the six
other tests demonstrated in Table 3. The advantage of OWC may be attributed to the
fact that it is an ultimately derived test after incorporating two kinds of burden tests and
two kinds of quadratic tests. Among the four gene-level test statistics in OWC, the score
test ( Ss) that we proposed is to find the appropriate weights for genetic variants which
allows the score statistic reaches its maximum. The power gained of OWC may be from
two types of maximization in our proposed method: 1) to find the appropriate weights
for the four gene-level tests in the combination to let the combination to reach its maxi-
mum; 2) to find the appropriate weights for genetic variants in the considered gene
to let Ss to reach its maximum. Therefore, the OWC test can reach the largest power.
When △ uses the settings of the fixed values, the power of the S2T and GATES methods
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 8 of 19

increases as the effect size increases (△ = (4,2,1) vs.(8,4,2) when A=(1,1,1), (1,1,− 1),
or (1,− 1,− 1)). When △ = (4,2,1), the powers of S2T and GATES are extremely low no
matter A = (1,1,1), (1,1,− 1), or (1,− 1,− 1). This implies that their powers may suffer
significant losses compared to the other methods when there are weak genetic effects.
S2T and GATES are all robust to the direction of effects among causal SNPs since S2T
is a quadratic method and GATES is a p-value combination method. When △ = (8,4,2),
the powers of S2T and GATES are high no matter A = (1,1,1), (1,1,− 1), or (1,− 1,− 1).
When one or two causal variants have weak protective effects and the other causal vari-
ant has medium risk effect, all of the methods are significantly less powerful except for
OWC (△ = (4,2,1) when A = (1,1,− 1), or (1,− 1,− 1)). This conclusion is verified by the
results from the normal distribution settings of △. The results of a uniform distribution
settings of △ confirm that the power of the burden test ST is attenuated when there are
different directions of effects of causal variants. Both AT and aSPU are adaptive methods
by combining the burden test and quadratic test methods together, suffering a relatively
small power loss when there are weak and different directions of effects. The power of a
method increases as the number of risk causal variants increases. For example, when we
keep two protective causal variants and increase the number of risk causal variants from
1, to 2, and then 3 (△ = (4,2,1) and A = (1,− 1,− 1), △ = (4,4,2,1) and A = (1,1,− 1,− 1),
△ = (4,4,4,2,1) and A = (1,1,1,− 1,− 1)), the power of all of the methods increases. sum-
STAAR is less powerful than the GATES method when △ uses the settings of the uni-
form and normal distributions but sumSTAAR is more powerful than GATES in some
of situations when △ uses the settings of the fixed values. In summary, our proposed test
OWC is robust and powerful regardless of whether the causal genetic variants in a gene
have the same or different directions of effects, especially when weak effect sizes exist.

Real data analysis

Schizophrenia GWAS summary data application
We further evaluated the performance of the proposed method OWC by applying it and
the other six methods to two SCZ summary datasets [22]. The two datasets were down-
loaded from the website of the Psychiatric Genomics Consortium (PGC) (URL https://​
www.​med.​unc.​edu/​pgc/​resul​ts-​and-​downl​oads). The first dataset is a SCZ meta analy-
sis GWAS dataset (13,833 cases and 18,310 controls), denoted as SCZ1 [23]. The sec-
ond dataset is a more recent study including 36,989 cases and 113,075 controls, denoted
as SCZ2 [24]. The MAF, estimated effect size, odds ratio, and p-value for 560,833 SNPs
on 17,866 genes are included in SCZ1. Similar information for 557,511 SNPs on 17,824
genes are included in SCZ2. Following Wu et al. [25], a gene was defined by including
all of the SNPs from 20 kb upstream to 20 kb downstream of the gene. Using OWC
and other six tests, we tested the association between the gene and the trait. The 1000
Genomes Project reference panel [18] was used to estimated the LD of pairwise SNPs
within each gene of the two datasets. To make fair comparisons among the seven tests,
we removed rare variants with MAF< 0.05 and kept one of a pair of SNPs with the coef-
ficient of pairwise LD r 2 > 0.5. After SNPs pruning in quality control, 174,648 SNPs on
17,467 genes in SCZ1 data and 174,275 SNPs on 17,420 genes in SCZ2 data are remained
in our final analysis. We used ­106 times of Monte Carlo simulation to estimate the
p-values for the OWC and aSPU method. The Bonferroni corrected significance level
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 9 of 19

Fig. 1 Venn diagram of the number of significant genes identified by OWC, aSPU, GATES, sumSTAAR and GW
for SCZ1

Fig. 2 Venn diagram of the number of significant genes identified by OWC, aSPU, GATES, sumSTAAR and GW
for SCZ2

≈ 2.80 × 10−6 was employed to claim significance in a genome-wide gene-based asso-

ciation study. We first conducted a GWAS for the SCZ1 data [20,899 individuals] with
the OWC and the other comparable methods to identify genes associated with SCZ. We
then detected genome-wide significant genes associated with SCZ based on the larger
SCZ2 dataset [150,064 individuals] which can be considered as a partial validation study
for the GWAS based on the SCZ1 data.
Figure 1 shows a Venn diagram of the number of significant genes identified in SCZ1
by the proposed method OWC, aSPU, GATES, sumSTAAR and GW. GW represents the
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 10 of 19

aggregation of genes identified by S2T, ST and AT. The OWC identified the most sig-
nificant genes (130 genes). sumSTAAR identified the least significant genes (45 genes).
Both aSPU and GW identified 92 significant genes. GATES identified 84 significant
genes. Among the 130 genes identified by OWC, 78 (i.e. 60%) contained genome-wide
significant SNPs (p-value 5 × ­10−8) within 20 kb in the SCZ1 data and 86 (around 66.2%)
contained genome-wide significant SNPs within 20 kb in the SCZ2 data. This offered sig-
nificant validation of the identified genes. Thus, our method identified more SCZ asso-
ciated genes than the other methods. More interestingly, OWC uniquely identified 49
genes in the SCZ1 data and 104 genes in the SCZ2 data. These genes were missed by
other methods. Among the 49 genes, 10 genes contained the genome-wide significant
SNPs within 20 kb in the SCZ2 data. These identified genes containing highly significant
SNPs gave credence to the power and validity of OWC. Overall, we identified 76 sig-
nificant and unique genes in the SCZ1 data with all these tests. Additional file 2: Signifi-
cant genes identified by OWC, aSPU, GATES, sumSTAAR, and GW in SCZ1 data, SCZ2
data, and UKB data. Tables S1 and S2 shows information about the significant genes
identified by OWC, aSPU, GATES, sumSTAAR and GW in SCZ1 data and SCZ2 data,
respectively.
Next, the seven tests were applied to the SCZ2 data. Figure 2 shows the numbers of
significant genes identified by OWC, aSPU, GATES, sumSTAAR and GW. Similarly, the
OWC identified the most significant genes (534 genes). Among the 534 genes, 398 genes
(74.5%) contained genome-wide significant SNPs (p-value 5 × ­10−8) within 20 kb in the
SCZ2 data. sumSTAAR identified the least significant genes (228 genes). GATES identi-
fied 432 significant genes. GW identified 431 significant genes, similarly, aSPU identified
445 genes. As expected, all the methords identified more significant genes in the SCZ2
data than in the SCZ1 data since the sample size of the SCZ2 dataset is much larger than
that of SCZ1 [22]. Again, our method OWC is more powerful than the other methods
in terms of the total number of significant genes being identified. We further noticed
that each of these tests identified some unique genes but missed by the others. This sug-
gests that different tests may be powerful in different scenarios. In the SCZ2 data, OWC
identified 104 significant and unique genes (Additional file 2: Significant genes identi-
fied by OWC, aSPU, GATES, sumSTAAR, and GW in SCZ1 data, SCZ2 data and UKB
data. Table S2 shows information about significant genes identified by OWC, aSPU,
GATES, sumSTAAR, and GW in SCZ2 data).
The computational time of OWC in a genome-wide association study is acceptable,
though the Monte Carlo simulation method is employed to estimate the p-value of
OWC. For example, there are 17,467 genes in the SCZ1 GWAS summary data. We used
­106 times of simulations to estimate the p-value of OWC. The computational time of
p-value estimation of OWC for a gene based on 1­ 06 simulations is about 20 minutes
when we use the R package of OWC with the fast algorithm [25] on a Dell PowerEdge
C6320 server which includes two 2.4 GHz Intel Xeon E5-2680 v4 fourteen-core pro-
cessors with average memory as 600 MB. The estimated time for completing a whole
genome-wide association study for the 17,467 genes would be less than a day if we run
the jobs on 500 such servers concurrently.
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 11 of 19

Fig. 3 Venn diagram of the number of significant genes identified by OWC, aSPU, GATES, sumSTAAR and GW
for UKB

T2D GWAS summary data application

Furthermore, we performed a comprehensive study for fasting glucose in a type 2 diabe-
tes (T2D) GWAS summary data obtained from the UK Biobank component of the Euro-
pean DIAMANTE study (denoted as UKB). It included over 440,000 individuals [19,119
cases and 423,698 controls] of European ancestry. This GWAS using the UK Biobank
Resource under Application Number 9161 (McCarthy) was restricted to HRC variants.
We downloaded the GWAS summary data from http://​www.​type2​diabe​tesge​netics.​org/​
infor​matio​nal/​data. The UKB summary data consists of information about MAF, esti-
mated effect size, odds ratio, and p-value for approximately 17,850 genes [27]. The same
filtering and analyzing procedure used in the SCZ data was employed in the UKB data.
The significance level 0.05/17, 850 ≈ 2.80 × 10−6 was used in this study. We performed
­106 simulations to estimate p-values for the OWC and aSPU method.
The Venn diagram in Fig. 3 shows the number of significant genes identified by OWC,
aSPU, GATES, sumSTAAR and GW, respectively. The OWC identified 236 significant
genes which is much larger than the number of genes identified by the other methods
(aSPU [182 genes], GATES [166 genes], sumSTAAR [82], and GW [179 genes]). Around
41.1% [97 out of 236] of the significant genes identified by OWC contained the genome-
wide significant SNPs (p-value < 5 × ­10−8) within 20 kb in the UKB data [27]. Based
on the number of significant genes identified in the UKB data, we can further conclude
that the proposed OWC method performed the best compared to the other tests. Addi-
tional file 2: Significant genes identified by OWC, aSPU, GATES, sumSTAAR, and GW
in SCZ1 data, SCZ2 data and UKB data. Table S3 shows the information about the sig-
nificant genes identified by all the methods in the UKB data.
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 12 of 19

Discussion
Weighting genetic variants in a gene appropriately plays an important role to boost
the power of a gene based association test. In this paper, we propose a novel combina-
tion test - OWC. This is a general linear combination test incorporating four different
weighting schemes: two constant weights and two weights proportional to normal sta-
tistics Z . The burden test, WSS, SSU, and score test are four typical gene-based tests,
which are included in the OWC as its special cases. When we focus on rare variants
analysis summary data, the elements on the diagonal of matrix A can be estimated from
the beta distribution with pre-specified shape parameters in its density function as 1 and
25. In this situation, the method SSU contained in OWC is the SKAT method. Therefore,
we can view the SKAT and SKAT-O methods as special cases of the proposed method.
When we have data from transcriptome-wide association studies, we can set the ele-
ments of the diagonal of matrix A being the estimated cis-effects from gene expression
as weights of variants for the WSS. Then, the WSS and SSU contained in the proposed
OWC method become PathSPU(1) and PathSPU(2) [28]. As a general method with a
maximized test statistic, the OWC can reach the largest power.
Furthermore, we show that the general linear combination test statistic can reach
its maximum when the weight is estimated as a certain value. For example, the score
test SS , as a special case of OWC, reaches its maximum when the weight is the prod-
uct of the inverse of the correlation matrix R among SNPs and Z-scores. A correct
estimation of the correlation matrix R is critical. To alleviate the errors in estimat-
ing R , Deng and Pan (2018) proposed an estimator of the correlation matrix R . Their
idea is similar to multiple imputation [29]. In real studies, we suggest to remove low
frequency (e.g. MAF < 0.05) variants and one of a pair of SNPs with pairwise LD r 2
greater than a prespecified threshold. We tested OWC on ten genes based on a real
SCZ1 data and the estimated ρ3 was always larger than 0.5. In this case, the score test
may make the main contribution in the power of OWC. When the correlation among
SNPs is ignored (i.e. R = I ), OWC becomes the SSU test. ST, S2T, AT, aSPU, GATES,
and sumSTAAR are the most popular existing methods using GWAS summary data.
We compared the performance of the proposed test OWC with the six comparison
methods in both simulation studies and real data analyses. Extensive simulation stud-
ies demonstrate that the proposed test OWC is not only valid but also powerful in
most of the scenarios. In real data analyses, OWC identifies the largest number of
disease associated genes compared to the other comparison methods.
True disease model is usually unknown. Disease models underlying different diseases
may be different: some of the disease models may include causal genetic variants with
same directions while other disease models may include causal genetic variants with dif-
ferent directions. In addition, some diseases models may include some weakly associ-
ated genetic variants, while other disease models may include some strongly associated
genetic variants. There is no uniformly most powerful test that is powerful in every situ-
ation. An association test may perform well in one dataset, but may perform less well
in another dataset. For example, SCZ can be considered as a representative of complex
disease. People have identified some common genetic variants with weak effects on SCZ.
These variants may be working in tandem to produce SCZ. A robust, flexible method
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 13 of 19

such as OWC can elucidate these weakly associated genetic variants better so that the
roles of theses genetic variants in disease etiology can be understood more clearly. The
proposed OWC method can be a useful tool as it adapts to the underlying biological dis-
ease model for a disease by selecting ρ based on the data.
In summary, the novelty of the proposed method lies in two aspects: 1) proposing a
new score test Ss which reaches its maximum through finding the certain weights for
genetic variants; 2) proposing a new combination method OWC which reaches its maxi-
mum through finding certain weights for the combination of the four component tests.
Also, the score test is a component of OWC. Through using two types of optimizations,
the OWC is more powerful than other comparison methods in most situations which is
demonstrated in Table 3 on the manuscript.
The proposed OWC method only needs the publicly available GWAS summary statis-
tics as input, without the need to access raw genotype and phenotype data. Researchers
will be able to identify more novel disease associated genes with OWC by utilizing pub-
licly available GWAS summary data. Novel disease associated genes can shed more light
onto underlying mechanism of diseases. In this paper, we focus on developing a power-
ful genetic associated test using single trait GWAS summary data. The proposed OWC
method can be easily extended to analyze GWAS summary data for multiple traits. We
have implemented OWC in an R package which is freely available at https://​github.​com/​
Xuexia-​Wang/​OWC-R-​packa​ge.

Methods
Expressing gene based methods with a weighted combination of Z‑scores
Consider a sample including n individuals with both genotype and phenotype data avail-
able in a genomics region (gene or pathway) with M genetic variants (e.g. SNPs). For the
ith individual, denote yi as the trait value which is either a quantitative or qualitative trait
′
(1 for cases and 0 for controls), denote Xi = (xi1 , . . . , xiM ) as the genotypic score for the
considered region, where xim ∈ {0, 1, 2} is the number of minor alleles at the mth variant.
xim can also be the number of minor alleles in dominant, recessive coding, or imputed
dosage that the ith individual has at the mth variant. Although the formulas derived in
the Methods section is based on genotypes with additive coding, the conclusions are still
held when the genotypes are centered with mean 0.
The generalized linear model was used to model the relationship between the trait and
the genetic variants in the considered region:
′
f (E(yi |Xi )) = β0 + βc Xi

where f (·) is a monotone “link” function and the vector βc is the parameter of inter-
est which represents the fixed effects of the genetic variants. Testing the association
between the genetic variants in the region and the trait is equivalent to test the effect of

the weighted combination of genetic variants gi = M m=1 wm xim on the trait. Under the
0

generalized linear model, we propose to use the score test statistic [30] to test the null
hypothesis H0 : βc = 0. The score statistic can be expressed as follows:
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 14 of 19

 n 2
− ȳ)(gi − ḡ)
i=1 (yi
S(w10 , . . . , wM
0
) = n n 2
n 2
i=1 (yi − ȳ) i=1 (gi − ḡ)

′
The score test statistic S can be viewed as a function of weight W0 = (w10 , . . . , wM 0 )
. Let
′ ′ ′
Y = (y1 , . . . , yn ) , X = (X1 , . . . , Xn ) . Denote P = In − n 1n 1n, where 1n represents a col-
1
′
umn vector containing all ones. P is an idempotent matrix. That is, P = P , PP = P . Con-
′
sidering xi = Xi W0, we can rewrite the score test as:
′ ′ ′
W0 X PYY PXW0
S(w10 , . . . , wM
0
)=n ′ ′ ′
W0 X PXY PY W0

Detailed derivation of the aforementioned score statistic can be found in the supple-
mentary materials (Additional file 1: Derivation of the score test). When real genotype
and phenotype data are available, the score statistic can be maximized and extended to
a General method to Test the effect of the Optimally Weighted combination of genetic
variants in a gene (G-TOW) [30].
To test the association between a trait and a genetic variant, a Z test is usually
employed. We can use the Z test below to test the main effect of the mthvariant in the
′
considered region on the trait. Zm = √Y PXm·
′ where σ = 1 ′
n Y PY and
σ Xm· PXm·
′
Xm· = (xm1 , . . . , xmn ) .
Denote the linkage disequilibrium (LD) matrix for the considered region as
′
R = diag(D)−1/2 Ddiag(D)−1/2, where D = X PX and diag(D) denotes the diagonal
matrix of D . When GWAS summary statistics such as the Z-scores and the LD matrix
for genetic variants in the considered region are available, the score statistic can be writ-
ten as:
′ ′
W ZZ W
S(w1 , . . . , wM ) = ′ (1)
W RW
′ ′
where Z = (Z1 , . . . , ZM ) and W = (w1 , . . . , wM ) = diag(D)1/2 W0 (see Additional file 1:
Derivation of the score test). From equation (1), the score statistic S is equivalent to a
linearly weighted test statistic based on Z-scores:


M
′
L(w1 , . . . , wM ) = wm Zm = W Z (2)
m=1

Under the null hypothesis, Z follows multivariate normal distribution with mean 0 and
covariance matrix R [31]. This conclusion clearly demonstrates that testing the weighted
combination of genetic variants in a considered region using the score test is the same as
using the weighted combination of Z-scores for those variants.
′
In the aforementioned weight function W = (w1 , . . . , wM ) , the true value of each
weight is unknown and must be determined biologically or empirically. Therefore, in real
data analysis, we should give reasonable values of weights in advance for a gene-based
test. If all or most of the genetic variants in the region have almost an equal effect size in
the same direction of association, we set wm = 1 for m = 1, . . . , M , and the test becomes
the burden test LB = L(1, . . . , 1), which sums up the association signals across all the
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 15 of 19

variants and obtains high power. If we believe that the causal genetic variants would be
subject to “purifying selection” and thus appear less frequently in the population than

neutral variants, we set wm = 1/ pm (1 − pm ) , where pm denotes MAF of the mth vari-
 
ant, and obtain LW = L(1/ p1 (1 − p1 ), . . . , 1/ pM (1 − pM )), which is the weighted
sum statistic (WSS) [12]. If we assume that the values of the weights W come from gene
expression or functional annotation data, the test degenerates into the PathSPU(1) test
[28]. We know that S(w1 , . . . , wM ) follows central chi-square distribution with 1 degree
of freedom (χ12) and L(w1 , . . . , wM ) follows multivariate normal distribution with mean 0
′
and covariance matrix W RW under the null hypothesis, given the choice of the weight
function W is not proportional to Z .
′
As a function of W = (w1 , . . . , wM ) , either the score test S(w1 , . . . , wM ) or the linear
weighted test statistic L(w1 , . . . , wM ) can reach its maximum when we choose an appro-
priate weight W . According to conclusions in Li and Lagakos [32], we have
 
L(w1 , . . . , wM )2
sup{S(w1 , . . . , wM )} = sup
W W Var(L(w1 , . . . , wM ))
 ′ ′

W ZZ W
= sup ′
W W RW
′
= Z R−1 Z

When W  = R−1 Z , the score test statistic S(w1 , . . . , wM ) reaches its maximum value.
Given the asymptotic null distribution of Z in Eq. (2), we define the score test
′
 Z = Z R−1 Z
SS = W
′
(3)

which follows central chi-square distribution with M degrees of freedom (χM 2 ). The

appropriate weights can be obtained when the linear weighted test statistic reaches
its maximum value [33]. Although SS may not have high power when its degree free-
dom is large, it gives higher weights to the SNPs that have weak correlation with
other SNPs. When the correlation matrix R of Z is a diagonal matrix denoted as
A = diag(a1 , . . . , aM ) where 0 < ai ≤ 1, that is, R = A, we have W = A−1 Z . The score
test in Equation (1), which is equivalent to the linear weighted test in Equation (2), will
reach its maximum value when W = A−1 Z .
To test the association between genetic variants in a considered region and a
trait, Kwak and Pan [6] proposed a class of approaches called sum of powered score
 γ
(SPU) tests along with its data-adaptive version (aSPU), SPU (γ ) = M m=1 Zm and
γ = 1, 2, . . . , 8, ∞. The SPU method can also be viewed as a special combination test
method with weight W = Z γ −1. aSPU can be viewed as a data-adaptive weighted com-
bination test method.
When the diagonal matrix A is the identity matrix A = I , we denote the test in
′
Equation (3) as SQ = Z Z , which is the same as the sum of squared score test (SSU)
[34] and the variance component test [35]. Based on the asymptotic null distribution
′
of Z in Equation (2), the test SQ = Z Z follows a mixture of chi square distribution

under the null hypothesis: SQ ∼ M m=1 m χ1 , where 1 , . . . , M are the eigenvalues of
2

R . Particularly, if we set the diagonal element of A as the beta distribution density

function with pre-specified shape parameters as 1 and 25, which are evaluated at the
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 16 of 19

corresponding sample MAF in the data, the score test degenerates into the sequence
kernel association test (SKAT) for rare variants [13]. If the value of the diagonal ele-
ments A comes from a set of gene expression derived weights, the score test degen-
erates into PathSPU(2) test method [28]. Naturally, these two methods (SKAT and
PathSPU(2)) both follow a mixture of chi square distribution under the null hypoth-
esis. In our paper, we only consider GWAS summary data for common variants, so
we set A as the identity matrix for this case.

A new gene‑based method

We have proved and demonstrated that most of the gene-based associate tests can
be expressed as a weighted combination of Z-scores. Thus, we can propose a new
weighted combination method by utilizing the good properties of different weights.
The statistics of LB , LW , SS , and SQ represent four typical weighted methods. To
combine the strength of LB , LW , SS , and SQ , we consider their weighted average:

Lρ = ρ1 (LB )2 + ρ2 (LW )2 + ρ3 SS + ρ4 SQ
′
= Z AZ
′ ′
where A = ρ1 11 + ρ2 W W + ρ3 R−1 + ρ4 I , 1 denotes a column vector containing all
1s, ρ1 + ρ2 + ρ3 + ρ4 = 1, and 0 ≤ ρi ≤ 1 for i = 1, 2, 3, 4 . Under the null hypothesis, for
a given ρ , Lρ is a linear combination of independent central χ12 random variables:


M
Lρ ∼ i χ12
i=1

where χ12 denotes a central χ 2 random variable with 1 degree of freedom and i for
i = 1, . . . , M are the eigenvalues of RA [4]. We propose a novel method - OWC. For a
set of values of ρ , OWC test can be achieved by using the minimum p-value across the
values of ρ:

T = min pLρ (4)

ρ

where pLρ is the estimated p-value of Lρ . Naturally, T can be obtained by a simple grid
search across a range of ρ : {ρ1 , ρ2 , ρ3 , ρ4 }. The test statistic T = min{pLρ1 , . . . , pLρ4 }. We
search over ρi ∈ (0, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1) for i = 1, 2, 3, 4 . Specifically, if
ρ2 = ρ3 = 0 in ρ , Lρ can be rewritten as ρ1 (LB )2 + (1 − ρ1 )SQ , which is equivalent to
SKAT-O test method [14].

p‑value estimation
Monte Carlo simulations are used to obtain the p-values for T in a single layer of
simulations. Briefly, after obtaining R , we first simulate null scores of Z (b) ∼ N (0, R)
for b = 1, . . . , B . Then, we use the null scores to calculate the null test statistic T b fol-
lowing the aforementioned procedure for each b, and then the p-value of the test is
the proportion of the number of the null test statistic T b with T b ≤ T [36]. A larger B
is needed to estimate a smaller p-value.
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 17 of 19

The aforementioned vector Z (b) can be generated in the following way [37]: we first
generate a vector L with M elements where each element is independently generated
from a standard univariate normal distribution with mean 0 and variance 1; that is,
L ∼ N (0, I). We then have Z (b) = DL , where D is obtained from Cholesky decompo-
′
sitions of R with R = DD . Specifically, for the test statistic T (Z, R) as a function of Z
and R , we can estimate its p-value in detail as follows:

1 Generate independent Z (b) ∼ N (0, R) for b = 1, . . . , B.

2 Using asymptotic distribution of Lρ under null hypothesis, calculate the null test sta-
tistic T by searching across a range of ρ for Z and Z (b), respectively.
3 Finally, the p-value for the T test, pT , is

B 
 (b)

pT = I T (Z , R) ≤ T (Z, R) + 1 /(B + 1) (5)
b=1

where T (Z, R) is the value of T test based on the observed data, T (Z (b) , R) is the
value of T test based on the bth sampling data.
If the Z statistic in the summary data is not provided, we need to first transform the
p-value in the summary data into a Z statistic using Z = sign(β)�−1 (1 − p/2), where
 is the cumulative distribution function of the standard univariate normal distribu-
tion. Then, a similar procedure can be used to obtain the p-value of the test T.
One limitation of the Monte Carlo simulation to estimate p-values, such as the
above one, is the computational burden. Especially, when there are about twenty
thousands genes in a GWAS and a small significance level is used to claim signifi-
cant findings. We adopted a fast algorithm [26] to estimate p-values, which will dra-
matically reduce the computational time. This algorithm reduces computational time
by scarifying the precision of the p-value estimation for those tests with large true
p-values.
We first define the following parameters for the algorithm:

Bmax = maximum number of random sampling (e.g.106)

B0 = minimum number of random sampling (e.g. 10)
p0 = a constant × significance level (e.g.5 × 10−6)
M = multiplying increment for the number of random sampling (e.g. 10)
The fast algorithm works as follows:

Step 0 Calculate the statistic T of OWC based on the observed data

Step 1 Set initial values: p0 = 10−5, Bmax = 106, B0 = 10, M = 10, B = B0
Step 2 Use Eqs. (4) and (5) to estimate p-value, p̂. Let B = B × M
Step 3 If p̂> p0 or B > Bmax , report p̂ and stop; otherwise go to step 2.

Conclusions
Current gene-based association tests, while providing greater interpretability and
power over usual single variant association tests, still have many limitations such
as weights predetermined biologically or empirically. In this paper, we propose a
Zhang et al. BMC Bioinformatics (2023) 24:2 Page 18 of 19

combination test OWC to overcome these limitations. OWC is a general linear com-
bination test which uses GWAS summary statistics as its input and incorporates dif-
ferent weighting schemes, and includes traditional gene-based tests as its special cases.
Simulation studies and real data analyses demonstrate that OWC is more powerful
than comparable methods in many scenarios and can adapt to the (generally unknown)
underlying genetic architecture of the trait of interest. While the focus of this paper was
single-trait analysis, OWC can be easily extended to analyze GWAS summary data for
multiple traits.

Supplementary Information
The online version contains supplementary material available at https://​doi.​org/​10.​1186/​s12859-​022-​05114-x.

Additional file 1. Linkage equilibrium matrix of gene EPB41.

Additional file 2. Significant genes identified by OWC, aSPU, GATES, sumSTAAR, and GW in SCZ1 data, SCZ2 data
and UKB data.

Acknowledgements
X. Wang was supported by the Florida International University startup fund. X. Wang was also supported by the Univer-
sity of North Texas Foundation which was contributed by Dr. Linda Truitt Creagh. The content is solely the responsibility
of the authors and does not necessarily represent the views of the University of North Texas Foundation and Dr. Linda
Truitt Creagh. X. Gao was supported by National Institutes of Health (NIH; Bethesda, MD, USA) grants R01EY027315 and
RF1AG060472. The content is solely the responsibility of the authors and does not necessarily represent the official views
of the National Institutes of Health. A superior high-performance computing infrastructure at University of North Texas,
was used in obtaining results presented in this publication.

Author Contributions
All authors read, reviewed, and approved the manuscript. X.W. designed and supervised the study. J.Z. and X.W. pro-
posed the statistical methods. J.Z., S.G.and X.W. performed simulation studies and real data analysis. S.G. and J.L. cleaned
the real data. X.L and X.G. helped in interpreting findings. J.Z., X.L., S.G.and X.W. wrote the manuscript.

Funding
This work is supported in part by funds from the National Institutes of Health (NIH; Bethesda, MD, USA) grants
R01EY027315 and RF1AG060472.

Availability of data and materials

The GWAS summary data of schizophrenia that was obtained from the Psychiatric Genomics Consortium can be
downloaded from https://​www.​med.​unc.​edu/​pgc/​downl​oad-​resul​ts/. The GWAS meta-analysis summary data for fasting
glucose that was obtained from the European DIAMANTE study can be downloaded from https://​t2d.​hugea​mp.​org/​
datas​ets.​html.

Declarations
Ethics approval and consent to participate
Not applicable.

Consent for publication

Not applicable.

Competing interests
The authors declare that they have no competing interests.

Received: 2 June 2022 Accepted: 13 December 2022

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