# Integrative Decomposition Procedure and Kappa  Statistics for  the Distinguished Single Molecular Network  Construction and Analysis

Authors: Lin Wang; Ying Sun; Minghu Jiang; Xiguang Zheng
Journal: Journal of Biomedicine and Biotechnology
 (2009)

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## Abstract

Our method concentrates on and constructs the distinguished single gene network. An integrated method was proposed based on linear programming and a decomposition procedure with integrated analysis of the significant function cluster using Kappa statistics and fuzzy heuristic clustering. We tested this method to identify ATF2 regulatory network module using data of 45 samples from the same GEO dataset. The results demonstrate the effectiveness of such integrated way in terms of developing novel prognostic markers and therapeutic targets.


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## Body

## 1. Introduction

In the postgenomic  era, with microarray technologies producing great deal of gene expression data, mining these data to
get insight into biological processes at system-wide level has become a
challenge for bioinformatics. On one hand, due to the complex and distribute
nature of biological research, there is a great deal of methods
for inferring gene regulatory networks. But all these methods focused on constructing
the complicated entire network calculated from the given microarray data. The tremendous
amounts of genes in those networks distribute analysts’ attention, so it is
hard to get any clear perception of valuable knowledge from such complicated networks,
let alone further study of each single gene. On the other hand, the wide spread
of knowledge over independent databases aggravates the hardness of integrating
comprehensive annotation information for genes and lowers the study effectiveness. Thus, a
novel method integrating both single molecular network construction and highly
centralized gene-functional-annotation analysis is in demand for gene network
and functional analysis.This paper proposed an integrated method based on linear programming
and a decomposition procedure with integrated analysis of the significant
function cluster using Kappa statistics and fuzzy heuristic clustering. Our
method concentrates on and constructs the distinguished single gene network
integrated with function prediction analysis by DAVID. For the distinguished
single molecular network, we did (1) control and experiment comparison,
(2) identification of activation and inhibition networks, (3) construction of upstream and
downstream feedback networks,
and (4) functional module construction. We tested this method to identify ATF2
regulation network module using data of 45 samples from one and the same GEO dataset. The results
demonstrate the effectiveness of such integrated way in terms of developing
novel prognostic markers and therapeutic targets.


## 2. Methods

### 2.1. Distinguished Single Molecular Network Construction

The entire network was constructed
using GRNInfer [1] and GVedit tools. GRNInfer is a novel mathematic method
called gene network reconstruction (GNR) tool based on linear programming and a
decomposition procedure that is used for inferring gene networks. The method
theoretically ensures the derivation of the most consistent network structure
with respect to all of the datasets, thereby not only significantly alleviating
the problem of data scarcity but also remarkably improving the reconstruction
reliability. The general solution for a single dataset is
the following (1), which
represents all of the possible networks:(1)J=(X′−A)UΛ−1VT+YVT=J^+YVT, where J=(Jij)n×n=∂f(x)/∂x is an n × n Jacobian matrix or
connectivity matrix, X=(x(t1),…,x(tm)), A=(a(t1),…,a(tm)), and X′=(x′(t1),…,x′(tm))
are all n × m matrices with xi′(tj)=[xi(tj+1)−xi(tj)]/[tj+1−tj]
for i=1,…,n; j=1,…,m. X(t)=(x1(t),…,xn(t))T∈Rn, a=(a1,…,an)T∈Rn, xi(t)
is the expression level (mRNA concentrations) of gene i at time instance t. y=(yij) is an n × n matrix,
where yij is zero if ej≠0 and is otherwise an
arbitrary scalar coefficient. ⋀−1=diag(1/ei)
and 1/e is set to be zero if ei=0. U is a
unitary m × n matrix of left eigenvectors, ⋀=diag(e1,…,en)
is a diagonal n × n matrix containing the n eigenvalues, and VT is the transpose of a unitary n × n matrix of
right eigenvectors.But the
entire network is too complex to get any clear perception of such complicated
relationships among those genes, let alone further study of each single gene. We
constructed the distinguished single molecular network by selecting the
centered gene and its directly related genes based on the entire network for
further study. We take into account the effectiveness of biology study in order
to concentrate on single molecular network rather than the intricate entire
network. It is helpful to get intensive and deep insight of the whole network. For
the distinguished single molecular network, we did (1) control and experiment
comparison, (2) identification of activation and inhibition networks, (3) construction
of upstream and downstream feedback networks, and (4) functional module construction.


### 2.2. Functional Annotation Clustering

For the function of genes that is neither
determined by their sequence nor by the protein families they belong to [2], the
function of those genes included in the same single molecular network should not
be interpreted separately, but should be analyzed together according to the
whole single molecular network. This method takes into account the network
nature of biological annotation contents in order to concentrate on the larger
biological picture rather than an individual gene. We used DAVID to do
functional annotation clustering. It changes functional annotation analysis
from term- or gene-centric to biological module-centric [2] in accordance with
our network analysis aim.The DAVID gene functional clustering
tool provides typical batch annotation and gene-GO term enrichment analysis for
highly throughput genes by classifying them into gene groups based on their annotation
term co-occurrence [3]. DAVID uses a novel algorithm to measure relationships
among the annotation terms based on the degrees of their coassociation genes to
group similar annotation contents from the same or different resources into
annotation groups. The grouping algorithm is based on the hypothesis that
similar annotations should have similar gene members. The functional annotation
clustering integrates the same techniques of Kappa statistics to measure the
degree of the common genes between two annotations, and fuzzy heuristic
clustering to classify the groups of similar annotations according kappa values
[4, 5]. The tool also allows observation of the internal relationships of the
clustered terms by comparing it to the typical linear, redundant term report,
over which similar annotation terms may be distributed among many other terms.


## 2.1. Distinguished Single Molecular Network Construction

The entire network was constructed
using GRNInfer [1] and GVedit tools. GRNInfer is a novel mathematic method
called gene network reconstruction (GNR) tool based on linear programming and a
decomposition procedure that is used for inferring gene networks. The method
theoretically ensures the derivation of the most consistent network structure
with respect to all of the datasets, thereby not only significantly alleviating
the problem of data scarcity but also remarkably improving the reconstruction
reliability. The general solution for a single dataset is
the following (1), which
represents all of the possible networks:(1)J=(X′−A)UΛ−1VT+YVT=J^+YVT, where J=(Jij)n×n=∂f(x)/∂x is an n × n Jacobian matrix or
connectivity matrix, X=(x(t1),…,x(tm)), A=(a(t1),…,a(tm)), and X′=(x′(t1),…,x′(tm))
are all n × m matrices with xi′(tj)=[xi(tj+1)−xi(tj)]/[tj+1−tj]
for i=1,…,n; j=1,…,m. X(t)=(x1(t),…,xn(t))T∈Rn, a=(a1,…,an)T∈Rn, xi(t)
is the expression level (mRNA concentrations) of gene i at time instance t. y=(yij) is an n × n matrix,
where yij is zero if ej≠0 and is otherwise an
arbitrary scalar coefficient. ⋀−1=diag(1/ei)
and 1/e is set to be zero if ei=0. U is a
unitary m × n matrix of left eigenvectors, ⋀=diag(e1,…,en)
is a diagonal n × n matrix containing the n eigenvalues, and VT is the transpose of a unitary n × n matrix of
right eigenvectors.But the
entire network is too complex to get any clear perception of such complicated
relationships among those genes, let alone further study of each single gene. We
constructed the distinguished single molecular network by selecting the
centered gene and its directly related genes based on the entire network for
further study. We take into account the effectiveness of biology study in order
to concentrate on single molecular network rather than the intricate entire
network. It is helpful to get intensive and deep insight of the whole network. For
the distinguished single molecular network, we did (1) control and experiment
comparison, (2) identification of activation and inhibition networks, (3) construction
of upstream and downstream feedback networks, and (4) functional module construction.


## 2.2. Functional Annotation Clustering

For the function of genes that is neither
determined by their sequence nor by the protein families they belong to [2], the
function of those genes included in the same single molecular network should not
be interpreted separately, but should be analyzed together according to the
whole single molecular network. This method takes into account the network
nature of biological annotation contents in order to concentrate on the larger
biological picture rather than an individual gene. We used DAVID to do
functional annotation clustering. It changes functional annotation analysis
from term- or gene-centric to biological module-centric [2] in accordance with
our network analysis aim.The DAVID gene functional clustering
tool provides typical batch annotation and gene-GO term enrichment analysis for
highly throughput genes by classifying them into gene groups based on their annotation
term co-occurrence [3]. DAVID uses a novel algorithm to measure relationships
among the annotation terms based on the degrees of their coassociation genes to
group similar annotation contents from the same or different resources into
annotation groups. The grouping algorithm is based on the hypothesis that
similar annotations should have similar gene members. The functional annotation
clustering integrates the same techniques of Kappa statistics to measure the
degree of the common genes between two annotations, and fuzzy heuristic
clustering to classify the groups of similar annotations according kappa values
[4, 5]. The tool also allows observation of the internal relationships of the
clustered terms by comparing it to the typical linear, redundant term report,
over which similar annotation terms may be distributed among many other terms.


## 3. Results and Discussion

We tested this method using microarrays containing 22215 genes in
40 MPM tumors and 5 normal pleural tissues from one and the same GEO datasets. We
identified potential tumor molecular markers and chose the top 51 significant
positive genes with normalization of log2, the minimum fold change = 3.5, delta = 1.59,
and a false-discovery rate of 0% using SAM [6]. We selected activating
transcription factor (ATF)-2 because it is one of the most distinguished genes
in MPM. It is a member of the ATF/cyclic AMP-responsive element binding protein
family of transcription factors.


### 3.1. Normal Tissues and Tumor Comparisons of Distinguished Single Molecular Network

We, respectively, constructed the interaction network of the above
51 genes in healthy tissues and that in tumor using GRNInfer [1] and GVedit
tools and selected the ATF2-centered downstream subnetworks. With comparison of
these ATF2-centered subnetworks, we can get a more clear perception of the
notable differences between normal tissues and tumor, as shown in Figure 1. It
appeared that ATF2 inhibits C11orf9, C18orf10, C20orf31, CALD1, CAMK2G, DDX3X, FALZ, GLS, GOLGA2, ID2, NME2, NMU,
NONO, PAWR, PLOD2, PSMF1, RBMS1, RIC8A,
RNF10, TEAD4, TIA1, TNPO1, unknown2, unknown3, WBSCR20C, and ZF in normal
tissues, as shown in Figure 1(a). It appeared that ATF2 inhibits C11orf9, C15orf5, C18orf10,
C20orf31, CAMK2G, CDR2,
DDX3X, FALZ, FLJ10707, GLS, GOLGA2, ID2, KRT18, LRRC1, NME2, NMU, NONO, NSUN5,
OBSL1_2, PLOD2, PLXNA1, PTOV1, RBMS1, RIC8A, RNASEH1, RNF10, TEAD4, TIA1, UCK2, USP11, and ZF,
while it activates
CALD1 and TFAP2C in tumor,
as shown in Figure 1(b).ATF2 downstream network in (a) normal tissue and (b) MPM tissue.
(a)(b)With comparison between the two results, notable differences can
be shown clearly in order to get further perception of pathological changes in
MPM. For example, ATF2 target genes appeared in ATF2
activation to CALD1, TFAP2C in
MPM, as only shown in Figure2(b). Caldesmon (CALD1) is a potential actomyosin regulatory protein
found in smooth muscle and nonmuscle cells [7]. Transcription factor AP2-gamma
(TFAP2C) is alternatively
titled AP2. Families of related transcription factors are often expressed in
the same cell lineages but at different times or sites in the developing
embryo. The AP2 family appears to regulate the expression of genes required for
development of tissues of ectodermal origin such as neural crest and skin [8].
AP2 may also be involved in the overexpression of c-erbB-2 in human breast cancer cells [9].(a) ATF2 upstream inhibition network of MPM; (b) ATF2 upstream activation
network of MPM.
(a)(b)


### 3.2. Identification of Activation and Inhibition Networks for the Distinguished Single Molecule

We also identified the activation and inhibition  networks, respectively,
in order  to simplify and intensify the analysis process. For example, in ATF2
upstream network of MPM, as shown in Figure2, it appeared that C11orf9, CDR2, FALZ, FLJ10534, FLJ10707, FLJ21816, GLS,
LRRC1, NMU, OBSL1, PAWR, PLXNA1, PTOV1, RNASEH1, TEAD4, TNPO1, TNRC5, USP11,
and ZF inhibit ATF2, as shown in  Figure 2(a),
whereas C18orf10,
DDX3X, GOLGA2, ID2, KRT18, KRT19, NONO, NSUN5, OBSL1_2, PLOD2, PSMF1, RBMS1,
REC8L1, RIC8A, RNF10, TFE3, TIA1, unknown1, unknown3,
WBSCR20B, and WBSCR20C activate
ATF2, as shown in  Figure 2(b).ATF2 upstream genes TFE3,
REC8L1 showed activation to ATF2. TFE3 is a member of the
helix-loop-helix family of transcription factors and binds to the mu-E3 motif
of the immunoglobulin heavy-chain enhancer and is  expressed in many cell types
[10]. Nakagawa et al. [11] identified TFE3 as a transactivator of metabolic
genes that are regulated through an E box in their promoters which led to
metabolic consequences such as activation of glycogen and protein synthesis,
but not lipogenesis, in liver [11]. REC8L1 is the human homolog of yeast Rec8, a meiosis-specific
phosphoprotein involved in recombination events [12]. Brar et al. (2006) showed
that phosphorylation of the cohesin subunit REC8 contributes to stepwise
cohesin removal [13].


### 3.3. Constructing Feedback Network of the Distinguished Single Upstream and Downstream Gene

We took into account the feedback relationship and setup ATF2 feedback network, as shown in Figure3. ATF2 target genes
appeared in ATF2 inhibition to CDR2, GLS, and USP11, consistently, its upstream
genes also appeared in CDR2, GLS, and USP11 inhibition to ATF2. CDR2 is also
called CDR62, where CDR means cerebellar degeneration-related. On Western blot
analysis of Purkinje cells and tumor tissue, the anti-Yo sera react with at
least 2 antigens, a major species of 62 kD called CDR62 and a minor species of
34 kD called CDR34 [14]. Sahai (1983) demonstrated phosphate-activated
glutaminase (GLS) in human platelets [15]. It is the major enzyme yielding
glutamate from glutamine. Significance of the enzyme derives from its possible
implication in behavior disturbances in which glutamate acts as a
neurotransmitter [16]. USP11 is also called UHX1. Swanson et al. (1996) cited
evidence indicating that ubiquitin hydrolases play a role in oncogenesis
(oncogenes and tumor suppressor gene products are degraded in
ubiquitin-dependent pathways) [17]. The relationship of ATF2 with CDR2, GLS,
and USP11 represents a negative feedback loop.Figure 3
ATF2
feedback subnetwork of MPM.


### 3.4. Functional Module Construction of the Distinguished Single Gene

According to ATF2 upstream network, we did DAVID analysis of
function cluster, respectively. The DAVID functional annotation clustering
results appeared that one ATF2 regulation network was identified as consisting
of the ATF2 upstream genes including RBMS1, RNASEH1, PTOV1, NONO, C11orf9,
PSMF1, TIA1, TEAD4, GLS, ID2, USP11, TNPO1, PAWR, PLOD2, and TFE3, as shown in
Figure4.Figure 4
One ATF2 upstream gene metabolic network including RBMS1, RNASEH1, PTOV1, NONO,
C11orf9, PSMF1, TIA1, TEAD4, GLS, ID2, USP11, TNPO1, PAWR, PLOD2, and TFE3.According to Figure2, it appeared that RBMS1, NONO, PSMF1, TIA1,
ID2, PLOD2, TFE3 activate
ATF2; whereas
RNASEH1, PTOV1, C11orf9, TEAD4, GLS, USP11, TNPO1, and PAWR inhibit ATF2.RBMS1, NONO, TIA1, ID2, and TFE3 enhance nucleoside, nucleotide,
and nucleic acid metabolism because RBMS1, NONO, TIA1, ID2, and TFE3 are
involved in these metabolism; PSMF1 activation to ATF2 means the increase of Acyl-CoA
metabolism and porphyrin metabolism; PLOD2 activation to ATF2 indicates the progress
of cholesterol metabolism and other protein metabolism, as shown in Figure5.Figure 5
Molecular function and biological process from DAVID.RNASEH1, PTOV1, and TEAD4 inhibition to ATF2 decreases nucleoside,
nucleotide, and nucleic acid metabolism mediated by the
three genes; C11orf9 inhibition to ATF2 means the
decline of polysaccharide metabolism, whereas GLS represents the weakness of amino
acid and cyclic nucleotides metabolism; USP11 inhibition to ATF2 indicates the
fall-off in protein metabolism and modification, whereas PAWR in glycogen
metabolism, as shown in Figure5.


## 3.1. Normal Tissues and Tumor Comparisons of Distinguished Single Molecular Network

We, respectively, constructed the interaction network of the above
51 genes in healthy tissues and that in tumor using GRNInfer [1] and GVedit
tools and selected the ATF2-centered downstream subnetworks. With comparison of
these ATF2-centered subnetworks, we can get a more clear perception of the
notable differences between normal tissues and tumor, as shown in Figure 1. It
appeared that ATF2 inhibits C11orf9, C18orf10, C20orf31, CALD1, CAMK2G, DDX3X, FALZ, GLS, GOLGA2, ID2, NME2, NMU,
NONO, PAWR, PLOD2, PSMF1, RBMS1, RIC8A,
RNF10, TEAD4, TIA1, TNPO1, unknown2, unknown3, WBSCR20C, and ZF in normal
tissues, as shown in Figure 1(a). It appeared that ATF2 inhibits C11orf9, C15orf5, C18orf10,
C20orf31, CAMK2G, CDR2,
DDX3X, FALZ, FLJ10707, GLS, GOLGA2, ID2, KRT18, LRRC1, NME2, NMU, NONO, NSUN5,
OBSL1_2, PLOD2, PLXNA1, PTOV1, RBMS1, RIC8A, RNASEH1, RNF10, TEAD4, TIA1, UCK2, USP11, and ZF,
while it activates
CALD1 and TFAP2C in tumor,
as shown in Figure 1(b).ATF2 downstream network in (a) normal tissue and (b) MPM tissue.
(a)(b)With comparison between the two results, notable differences can
be shown clearly in order to get further perception of pathological changes in
MPM. For example, ATF2 target genes appeared in ATF2
activation to CALD1, TFAP2C in
MPM, as only shown in Figure2(b). Caldesmon (CALD1) is a potential actomyosin regulatory protein
found in smooth muscle and nonmuscle cells [7]. Transcription factor AP2-gamma
(TFAP2C) is alternatively
titled AP2. Families of related transcription factors are often expressed in
the same cell lineages but at different times or sites in the developing
embryo. The AP2 family appears to regulate the expression of genes required for
development of tissues of ectodermal origin such as neural crest and skin [8].
AP2 may also be involved in the overexpression of c-erbB-2 in human breast cancer cells [9].(a) ATF2 upstream inhibition network of MPM; (b) ATF2 upstream activation
network of MPM.
(a)(b)


## 3.2. Identification of Activation and Inhibition Networks for the Distinguished Single Molecule

We also identified the activation and inhibition  networks, respectively,
in order  to simplify and intensify the analysis process. For example, in ATF2
upstream network of MPM, as shown in Figure2, it appeared that C11orf9, CDR2, FALZ, FLJ10534, FLJ10707, FLJ21816, GLS,
LRRC1, NMU, OBSL1, PAWR, PLXNA1, PTOV1, RNASEH1, TEAD4, TNPO1, TNRC5, USP11,
and ZF inhibit ATF2, as shown in  Figure 2(a),
whereas C18orf10,
DDX3X, GOLGA2, ID2, KRT18, KRT19, NONO, NSUN5, OBSL1_2, PLOD2, PSMF1, RBMS1,
REC8L1, RIC8A, RNF10, TFE3, TIA1, unknown1, unknown3,
WBSCR20B, and WBSCR20C activate
ATF2, as shown in  Figure 2(b).ATF2 upstream genes TFE3,
REC8L1 showed activation to ATF2. TFE3 is a member of the
helix-loop-helix family of transcription factors and binds to the mu-E3 motif
of the immunoglobulin heavy-chain enhancer and is  expressed in many cell types
[10]. Nakagawa et al. [11] identified TFE3 as a transactivator of metabolic
genes that are regulated through an E box in their promoters which led to
metabolic consequences such as activation of glycogen and protein synthesis,
but not lipogenesis, in liver [11]. REC8L1 is the human homolog of yeast Rec8, a meiosis-specific
phosphoprotein involved in recombination events [12]. Brar et al. (2006) showed
that phosphorylation of the cohesin subunit REC8 contributes to stepwise
cohesin removal [13].


## 3.3. Constructing Feedback Network of the Distinguished Single Upstream and Downstream Gene

We took into account the feedback relationship and setup ATF2 feedback network, as shown in Figure3. ATF2 target genes
appeared in ATF2 inhibition to CDR2, GLS, and USP11, consistently, its upstream
genes also appeared in CDR2, GLS, and USP11 inhibition to ATF2. CDR2 is also
called CDR62, where CDR means cerebellar degeneration-related. On Western blot
analysis of Purkinje cells and tumor tissue, the anti-Yo sera react with at
least 2 antigens, a major species of 62 kD called CDR62 and a minor species of
34 kD called CDR34 [14]. Sahai (1983) demonstrated phosphate-activated
glutaminase (GLS) in human platelets [15]. It is the major enzyme yielding
glutamate from glutamine. Significance of the enzyme derives from its possible
implication in behavior disturbances in which glutamate acts as a
neurotransmitter [16]. USP11 is also called UHX1. Swanson et al. (1996) cited
evidence indicating that ubiquitin hydrolases play a role in oncogenesis
(oncogenes and tumor suppressor gene products are degraded in
ubiquitin-dependent pathways) [17]. The relationship of ATF2 with CDR2, GLS,
and USP11 represents a negative feedback loop.Figure 3
ATF2
feedback subnetwork of MPM.


## 3.4. Functional Module Construction of the Distinguished Single Gene

According to ATF2 upstream network, we did DAVID analysis of
function cluster, respectively. The DAVID functional annotation clustering
results appeared that one ATF2 regulation network was identified as consisting
of the ATF2 upstream genes including RBMS1, RNASEH1, PTOV1, NONO, C11orf9,
PSMF1, TIA1, TEAD4, GLS, ID2, USP11, TNPO1, PAWR, PLOD2, and TFE3, as shown in
Figure4.Figure 4
One ATF2 upstream gene metabolic network including RBMS1, RNASEH1, PTOV1, NONO,
C11orf9, PSMF1, TIA1, TEAD4, GLS, ID2, USP11, TNPO1, PAWR, PLOD2, and TFE3.According to Figure2, it appeared that RBMS1, NONO, PSMF1, TIA1,
ID2, PLOD2, TFE3 activate
ATF2; whereas
RNASEH1, PTOV1, C11orf9, TEAD4, GLS, USP11, TNPO1, and PAWR inhibit ATF2.RBMS1, NONO, TIA1, ID2, and TFE3 enhance nucleoside, nucleotide,
and nucleic acid metabolism because RBMS1, NONO, TIA1, ID2, and TFE3 are
involved in these metabolism; PSMF1 activation to ATF2 means the increase of Acyl-CoA
metabolism and porphyrin metabolism; PLOD2 activation to ATF2 indicates the progress
of cholesterol metabolism and other protein metabolism, as shown in Figure5.Figure 5
Molecular function and biological process from DAVID.RNASEH1, PTOV1, and TEAD4 inhibition to ATF2 decreases nucleoside,
nucleotide, and nucleic acid metabolism mediated by the
three genes; C11orf9 inhibition to ATF2 means the
decline of polysaccharide metabolism, whereas GLS represents the weakness of amino
acid and cyclic nucleotides metabolism; USP11 inhibition to ATF2 indicates the
fall-off in protein metabolism and modification, whereas PAWR in glycogen
metabolism, as shown in Figure5.


## 4. Conclusions

Our method concentrates on and constructs the distinguished single
gene network integrated with function prediction analysis by DAVID. For the
distinguished single molecular network, we did (1) control and experiment
comparison, (2) identification of activation and inhibition networks, (3) construction
of upstream and downstream feedback networks, and (4) functional module construction. We tested this
method to identify ATF2 regulation network module using data of 45 samples from
one and the
same GEO dataset. The results demonstrate the
effectiveness of such integrated way in terms of developing novel prognostic
markers and therapeutic targets.



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*Source: 726728-2009-04-14.xml*