Integrating GO annotations with expression data to
analyze microarray experiments
Pedro Carmona-Sáez 1, Mónica Chagoyen1, Andrés Rodríguez 2, Oswaldo Trelles2,
José María Carazo1 and Alberto Pascual-Montano1*
Abstract. The use of biological knowledge about genes and gene
products in microarray data analysis is a necessary task when the
aim is to discover the biological patterns hidden in gene expression
profiles. In this paper we present a data mining approach able to
integrate different data sources in order to extract associations
among gene characteristics and expression patterns. An
implementation of this method is included in the EngeneTM
software package [1], which is freely accessible upon request at
http://www.engene.cnb.uam.es.
1. INTRODUCTION
Microarray technique has become a very popular method able to
measure expression levels of thousands of genes or whole genomes
in a single experiment. In this way, large gene expression datasets
containing expression data of thousands of genes across tens or
hundreds of different experimental conditions are being generated.
In the analysis of microarray data a critical issue is to discover
biological patterns from gene expression profiles. Several research
groups have approached this problem using clustering techniques
to group genes that share similar expression profiles. In this way,
co-clustered genes can be examined in order to discover biological
connections among them, for example, common upstream
sequence motifs [2]. Identifying biological relationships that may
exist among large sets of genes is not a trivial task. In this context
different bioinformatics tools have been developed in order to
connect lists of genes to biological databases and extract the most
relevant biological themes [3-7]. Nevertheless, it is also frequent
that the researcher annotates co-clustered genes one by one using
biological databases or literature searches [7]. In this way,
expression data is independently analyzed and biological
knowledge is introduced to the analysis as a subsequent step.
Although this is a widely and very often successful strategy it has
some drawbacks [8]. Genes sharing similar expression levels, and
grouped into the same cluster, might not be involved in the same
biological process or, in the same way, genes with different
expression profiles can be functionally related. In addition,
grouping genes into separate and not related clusters does not take
into account the fact that many gene products participate in more
than one biological process. In general, the quality of clusters and
their ability to explain biological function can vary greatly [9]. A
challenge in this field is to develop methods able to integrate
biological knowledge with expression data.
In this paper we show the application of a non supervised data
mining method, Association rules Discovery technique (ARD), in
order to extract intrinsic associations among biological properties
of genes and expression patterns.
1 BioComputing Unit, Centro Nacional de Biotecnología (CNB-CSIC).
Cantoblanco, 28049 Madrid, Spain.
2 Computer Architecture Department, Universidad de Málaga, 29080,
Málaga, Spain.
* To whom correspondence should be addressed: pascual@cnb.uam.es
With this purpose, we have used the “biological process” category
of Gene Ontology as gene attributes. Gene Ontology (GO)
database is one of the most used information sources to categorize
and annotate gene products. Indeed, in the last few years, different
approaches have showed the great potential of GO annotations to
analyze gene expression datasets [5, 10-14]. The aim of this work
is to show an implementation of ARD able to extract relevant
associations among GO annotations and expression data. An
interesting property of the method is that each gene can be
annotated with several terms and all of them will be taken into
account to extract those associations that are frequent and highly
related based on user specified thresholds.
2. METHODS
2.1 Association Rules Discovery and transaction
database for microarray data
Association Rules Discovery is a data mining technique oriented
towards finding interesting associations or correlation relationships
among a large set of data. For example, to identify sets of attribute
values (items) that frequently occur together in the same
transaction, and then formulate rules that characterize these
relationships. An intuitive definition of the rules is given as follow:
Making a formal statement of the problem, let
I = {i1 , i2 ,..., in } be a set of literals called items. Let S be a set
of transactions, where each transaction T is a set of items such that
T ⊆ I . We can now say that a transaction T contains a set X of
X ⊆ T . An association rule is an implication of the
items in I if
form X → Y , where X ⊆ I , Y ⊆ I and X ∩ Y = φ . The
rule X → Y has support s if s% of the transactions in S contain
X ∪ Y . The left hand side of the rule is called antecedent and the
right hand side is called consequent. Such rules are usually
interpreted as follow: when X occurs, it is often the case that Y also
occurs in the same transaction.
Different measures can be used to point out the relevance of a rule.
The most important ones are: support (previously defined),
confidence and improvement. The confidence of the rule is defined
P( X ∪ Y )
as; , and it is the probability for a transaction which
P( X )
contains X to also contain Y. Support and confidence are the most
common and, in many cases the only parameters used to mine
meaningful association rules. However, it is important to note that
sometimes both of these measures are high, indicating an
association which could be good, and yet still produce a rule that is
not useful. This is the case in which the elements of the consequent
are very frequent in the transaction database. For example, consider
the following rule: {A → C, B} with support = 60% and
confidence = 80%. This rule indicates that in 80% of the
transactions containing A, B and C are also present. The above rule
looks like a good rule but, is it really a relevant association if B and
C are present in 100% of the total transactions? Thus, a third
measure of the quality of the rule is needed: the improvement.
P( X ∪ Y )
Improvement can be defined as; . Any rule with an
P( X ) P(Y )
improvement less than 1 does not indicate a real correlation
between antecedent and consequent. On the contrary, when
improvement is greater than 1 the resulting rule is better at
predicting the consequent.
Given a set of transactions S, the problem of mining association
rules is to generate all associations that have support greater than
the user-specified minimum support. The first and key step in the
generation of association rules is the mining of frequent items on
the database. Once the frequent items are located, the subsequent
rules can be formed straightforwardly among them [15, 16].
To effectively generate association rules based on all possible
combinations of items, Agrawal proposed the Apriori algorithm
[15]. The rationale of this method is to reduce the number of
frequent candidate items used for creating the rules by eliminating
those that do not satisfy a minimum frequency constraint. Apriori
algorithm and its many improved variants use a breadth first
approach for the exploration of the frequent-pattern search space,
which makes them inefficient when dealing with dense datasets,
loads of frequent items, long patterns or very low support
thresholds. On the other hand, recent depth first approaches [17],
show limitations in dealing with large sparse databases.
In addition to the exploration approach (breadth or depth first),
counting the frequency of items requires choosing a method and
data structure for projecting the transactions of the database onto
the representation of the frequent items search space. As it can be
gathered from literature [18] no single combination of exploration
approach and projection method is good for all situations, so it
would be necessary to select the correct algorithm implementation
to maximize efficiency for the particular application case.
In this work, we have developed our own association rule
discovery algorithm, a solution especially suitable for data
collections in which elements appear clustered in sparse but
strongly related groups as is the case with gene expression data.
The algorithm is efficient, in particular when searching for low-
support or rare data patterns, in which the explosive growth of the
search space can make its exploration extremely expensive in
memory and CPU requirements.
Our method for mining frequent patterns is based on breadth first
approach, but the breadth extension is limited to the items that are
actually present in the database [19] It directly uses the content of
the database (the transactions) to drive the search procedure,
performing an on-the-fly projection, enabling a memory and CPU
efficient exploration of rare and infrequent association rules, which
can have high levels of confidence and be of great interest for the
researcher.
What exactly constitutes an item or a transaction depends on the
application. To extract the type of rules that we propose in this
work a microarray dataset is transformed in a transaction database
in which genes represent transactions and the set of experiments in
which each gene is over- or under-expressed represent the itemset.
In this transaction database gene attributes can also be included as
items (see Table 1) and association rules on the form of
{Characteristic X→ [+]Exp.A, [+]Exp.B, [-]Exp. C} can be
extracted. These rules are read as ”most of the genes annotated
with characteristic X were over-expressed in experimental
condition A and B and under-expressed in experiment C”.
To construct the transaction database from microarray data the
expression matrix has to be transformed into a Boolean matrix. For
this purpose we can use statistical methods to detect differentially
expressed genes or simply use a threshold value [16, 20]. In this
particular application, we have used two expression thresholds.
Genes with expression value greater than the positive threshold
were considered as expressed, in the same way, genes with
expression value lower than the negative threshold were being
inhibited. Values between these two ranks were neither expressed
nor inhibited.
Table 1 Example of transaction database used to extract association rules
among gene attributes and expression patterns.
Transaction Itemset
Gene 1 [+]Exp.A, [+] Exp. B, [-]Exp.C, characteristic 1, characteristic 2
Gene 2 [+]Exp.B, [-]Exp.C, characteristic 3, characteristic 4
Gene 3 [+]Exp.A, [+] Exp.B, [-]Exp.C, [+]Exp.D, characteristic 1, characteristic 3
Gene 4 [-]Exp.A, [-]Exp.B, [-]Exp.C, characteristic 6
Gene 5 [+]Exp.A, [+]Exp.B, [-]Exp.C, characteristic 1, characteristic 4
Gene 6 [+]Exp.A, [+]Exp.B, [-]Exp.C, characteristic 1, characteristic 7
2.2 Filtering rules
One of the main drawbacks related to the extraction of Association
Rules is the huge amount of associations generated, and the fact
that many of the rules extracted contain redundant information. We
applied different filter options to process the obtained rules.
- Redundant consequent filter: We consider a rule to be redundant
if its consequent is contained in the consequent of another rule with
the same antecedent and with equal or higher values for support,
confidence and improvement. For example: for the association
rules X: {A→ C, D, F}, Y: {A → D} and Z: {A→ D, F}; Y and Z
are redundant if their values for support, confidence and
improvement are less or equal than the corresponding values of X.
- GO filter: This filter eliminates those rules that have the same
values for support, confidence and improvement and the same
consequent, but their antecedent contains parent terms in the GO
hierarchy. For example, for the two rules X: {G2 phase of mitotic
cell cycle → A} and Y:{cell cycle → A} with the same support,
confidence and improvement, Y is filtered out because the GO
annotation contained by X, “G2 phase of mitotic cell cycle”, is
more specific than the “cell cycle” term.
- Single antecedent filter: This option filters out all rules whose
antecedent contains more than one item. It is applied to extract
independent information about different biological processes.
2.3 Statistical significance of extracted rules
Although previously described support and improvement values
provide information about the association between the antecedent
and consequent parts of the rule, they do not inform about their
statistical significance [21]. The statistical significance of a rule
was evaluated here using the influence of statistical dependency
between the antecedent and the consequent of the rule. In order to
calculate the statistical significance, we used the χ2- test for
statistical independence[21, 22]
A p-value associated to each rule is computed under the
assumption that the null hypothesis of the test is true (both the
antecedent and consequent part of the rule are independent). For a
p-value of 0.01 we can reject the null hypothesis with a 99% of
confidence. In our case, all rules that passed the filter conditions
are statistical significant according to this test.
2.4 Expression dataset and annotation of GO terms
Iyer et al. monitored mRNA levels of human fibroblasts in
response to serum in a time course experiment. This serum
stimulation dataset [23] is publicly available at http://genome-
www.stanford.edu/serum/. Threshold values of 1 for over-
expression and -1 for down-expression were selected to generate
the transaction dataset. A value between –1 and 1 is neither up nor
down. Experiments name represent time in which sample was
harvested after serum exposure. Missing values were filled by k-
nearest neighbours approach with k = 10 [24] and mean expression
levels were calculated for replicates samples.
Biological processes associated to each gene was annotated based
on the controlled vocabulary of the Gene Ontology Consortium
(GO) [25]. For each gene we included all annotations for biological
process category. Moreover, it was done at different levels of the
GO hierarchy (level 3, 4 and 5) using annotations obtained with the
DAVID program [3].
3. RESULTS
To test the potential of ARD in extracting significant biological
associations from gene expression data we applied it to the serum
stimulation dataset reported by Iyer et al. [23]. Genes from this
dataset were annotated with their associated GO terms. We have
focused our attention on finding rules among biological processes
and expression patterns using the “biological process” category of
the GO ontology. The Gene Ontology Consortium has developed a
standardized and dynamic vocabulary about gene products in
several organisms at three different categories; Molecular
Function, Biological Process and Cellular Component [25]. The
three GO categories are hierarchy structured. At low levels in the
hierarchy the functional annotations are more specific but the
number of annotated genes decreases. Moreover, one gene can be
annotated with different GO terms at different levels of GO
hierarchy.
This hierarchical nature of the GO ontology and the fact that the
same gene can be annotated into different categories implies a
problem if only one GO term is considered for each gene [26]. For
example, a particular gene could be annotated into the “fibroblast
proliferation” category (GO:0048144) and another one into “cell
proliferation” (GO:0008283), which is the immediately parent
category. Obviously, these two genes are functionally related, but
essentially they have a different annotation. Moreover, one gene
can be annotated with different terms such as p21(WAF1), that is
involved in induction of apoptosis and regulation of cell cycle. Due
to each term is considered as a single item ARD allow users to
annotate with several attributes each gene and all will be taken into
account to discover associations. Association rules will be
extracted about those terms that appear in a significant number of
co-expressed genes and are highly associated to their respective
expression pattern. We used all annotations included for each gene
at different levels of the GO hierarchy using the DAVID program
[3]. 247 genes of the 517 were associated to at least one GO term.
Most of the genes were annotated with several terms of the
“biological process” category, for example ATP citrate lyase
(ACLY) has more than 27 annotations in this category including all
parental terms. We specified a minimum absolute support value of
4, a minimum confidence of 70% and a minimum improvement of
1. The ARD algorithm applied to this dataset was able to find more
than 19000 association rules. To verify that extracted rules reveal
associations are not likely to have occurred by chance gene
expression profiles and experiment expression profiles were
random permutated. Only two associations were extracted with
only one element in the consequent and the lowest improvement
value.
We applied the filtering criteria described in the methods section to
eliminate those rules containing a redundant consequent (redundant
consequent filter), more than one element in antecedent (single
antecedent filter) and some redundancy based on GO hierarchy
(GO filter). We selected only those rules with one item in the
antecedent because we were mainly focused on showing
information related to each one of biological processes obtained
from GO ontology. Nevertheless, item combinations in the
antecedent can provide information about co-occurrences of gene
characteristics in sets of co-expressed genes which can be very
interesting in some cases. For example, when transcription factors
that bind to promoters regions are used as gene attributes,
information about their co-occurrences is very important due to
different transcription factors can co-operate to regulate gene
expression. 29 associations remained after post-processing the
obtained rules (Table 2).

Table 2 Association rules generated using biological process from Gene Ontology. Consequent element is graphically represented
by coloured squares. Dark grey represents over-expression, light grey under-expression and empty squares represent neither over-
expression nor under-expression.
15m
30m

12h
16h
20h
24h
1h
2h
4h
6h
8h

Antecedent Confidence Support Improvement p-value

alcohol metabolism 80.00 3.24 12.35 5.25E-22
angiogenesis 100.00 2.02 5.74 8.63E-07
angiogenesis 80.00 1.62 6.18 6.48E-06
angiogenesis 80.00 1.62 5.34 3.86E-05
blood coagulation 71.43 2.02 4.77 2.18E-05
chemotaxis 100.00 2.43 9.15 1.46E-12
cholesterol metabolism 100.00 2.43 15.44 4.41E-21
cholesterol metabolism 83.33 2.02 15.83 4.32E-18
cytokinesis 87.50 2.83 4.24 2.05E-06
cytoplasm organization and biogenesis 80.00 3.24 3.87 2.21E-06
DNA replication 83.33 2.02 4.04 1.23E-04
DNA replication and chromosome cycle 90.91 4.05 4.01 3.21E-08
DNA replication and chromosome cycle 81.82 3.64 3.96 2.93E-07
electron transport 75.00 2.43 2.41 6.52E-03
inflammatory response 83.33 2.02 5.56 2.04E-06
lipid biosynthesis 75.00 3.64 2.41 7.79E-04
M phase 75.00 4.86 3.31 2.35E-07
mitotic cell cycle 70.37 7.69 3.10 3.56E-10
response to chemical substance 88.89 3.24 6.86 4.83E-12
response to chemical substance 77.78 2.83 7.12 5.86E-11
steroid biosynthesis 87.50 2.83 13.51 2.93E-21
steroid biosynthesis 100.00 3.24 3.21 1.93E-05
steroid metabolism 72.73 3.24 11.23 6.68E-20
steroid metabolism 81.82 3.64 7.48 1.27E-14
steroid metabolism 90.91 4.05 2.92 1.21E-05
sterol biosynthesis 100.00 2.83 15.44 2.01E-24
sterol biosynthesis 71.43 2.02 13.57 1.82E-15
sterol metabolism 100.00 3.24 15.44 8.71E-28
sterol metabolism 75.00 2.43 14.25 2.71E-19
To evaluate the biological significance of the generated
associations, we paid attention to the support and confidence
values assigned to each rule. The support of the rule indicates the
number of transactions (genes) in which the coincidence of a
specific process with the co-expression pattern occurs. Confidence
value represents the percentage of genes annotated into a given
category (antecedent) which have the expression pattern that
appears in the consequent of the rule. In this kind of rules, perhaps
confidence is the most significant value from the biological point
of view. For example, if only a small set of genes are annotated
within a given process in the entire dataset, the support value of
rules containing this process will be quite low but, if these rules
have a high confidence value they are telling us that most of the
genes annotated for this process show a similar expression pattern.
Automatically extracted rules confirm previous evidences of
biological processes associated to serum response [23, 27].
Looking at the obtained rules in table 2 we can see that most of the
genes involved in inflammatory response, blood coagulation and
angiogenesis were mainly over-expressed as shown in the
consequent element of the rules. These processes are well
characterized as serum response associated phenomena as well as
induction of genes involved in cell proliferation and cell cycle.
Extracted rules reveal that genes related with these last processes
were mainly over-expressed at last time points of the time course
experiment. Moreover, these rules reveals that over-expressed
genes are related to M-phase associated processes which is in
agreement with the observation of Iyer et al. that the onset of M
phase were around 16 h after serum stimulation.
In the original work Iyer et al. manually analyzed the genes
associated to each cluster in order to discover common biological
properties. They noted that genes involved in cholesterol
biosynthesis were under-expressed after serum exposure due to
serum provided cholesterol to the cell. This fact is also manifested
by extracted rules. We can see that 100% of genes specifically
involved in cholesterol metabolism were under-expressed at 12, 16
and 20 h after serum exposure.
Results obtained in our approach showed that this methodology can
be a useful tool to analyze gene expression data integrating GO
annotations and expression patterns. It is important to remark that
although ARD has been previously applied to microarray data
analysis [16, 20] the type of rules proposed and the purpose of the
application were totally different with respect to our approach.
Previous works have used ARD to extract association rules among
genes basing only on expression data on the form of {[+]Gene A→
[+]Gene B, [-] Gene C}, mining that in a significant number of
experiments Gene A and B are over-expressed and Gene C under-
expressed, and also that when Gene A is over-expressed is
probably that Gene B is also over-expressed and Gene C under-
expressed.
Information about gene ontology hierarchy was compiled without
selection of a unique level of the hierarchy which is an interesting
property of the method. Associations were extracted in an
automatic way without any pre-established assumption and
revealed important biological features of serum response based on
GO annotations. Once genes were annotated with all GO terms
associated to each one, the process of extracting association rules
was an easy and fast task. In this way, the researcher can focus on
analyzing associations that are expected to be significant, without
wasting time testing different gene attributes that are not highly
associated with co-expression patterns.
ACKNOWLEDGMENTS
This work was supported in part by the “Comisión Interministerial
de Ciencia y Tecnología” through grant CICYT (BIO2001-1237),
by the “Comunidad de Madrid” through grant 07B/0032/2002 and
by the N.I.H. through grant 1R01HL67465-01. This work has also
been partly funded by the project TEMBLOR (The European
Molecular Biology Linked Original Resources) through grant EU
(QLRI-CT-2001-00015) and FISBIO2002-10855-E. P.C.S. is the
recipient of a fellowship from Comunidad de Madrid (CAM).
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