C h a p t e r
10
Integrative Systems Biology:
Implications for the
Understanding of Human
Disease
M. Michael Barmada
“What is now proved was once only imagined.”
William Blake
“All models are wrong, but some are useful.”
George Box [1]
INTRODUCTION
From the smallest collections of elementary particles
to the largest collections of galaxies, all matter is
constructed of interacting collections of elements, or
networks. Interactions within and between these net-
works give rise to the observable properties of matter.
Biological networks are no exception, from the level
of interacting enzymes and substrates which form a
pathway, up to the level of the biosphere, incorporat-
ing all living things and their relationships. Networks
have recently generated considerable interest in
biological sciences, not because of any change in our
basic knowledge about them, but because of key tech-
nological changes that have enhanced our ability to
interrogate them and thereby develop descriptive
models allowing prediction of their behavior. This
shift in ability has fueled new perspectives on how to
apply the scientific method to biomedical science,
emphasizing integration instead of reduction. Systems
(or network) biology focuses on the investigation of
complex interactions in biological systems. Much of
the promise of systems biology in biomedicine revolves
around its potential to explain disease as a perturba-
tion of a normal system. Many disease syndromes
include elements from multiple tissue-specific and sys-
temic systems. For example, chronic pancreatitis is
Molecular Pathology # 2009, Elsevier, Inc. All Rights Reserved.
n David C. Whitcomb
defined by variable amounts of maldigestion (loss of
pancreatic acinar cell function), diminished bicarbon-
ate secretion (loss of duct cell function), diabetes
mellitus (loss of islet cell function), inflammation
(immune system), fibrosis (regenerative systems), pain
(nervous system), and cancer risk (DNA repair and cell
cycle regulation systems). Since genetic and environ-
mental factors affect each of these systems in different
ways, it is not surprising that dysfunction of these sys-
tems in response to stress or injury will be variable
between patients. However, it is also clear that each
of these systems interacts with each other. Further-
more, for the systemic systems the commonly dysfunc-
tional components of effector or regulatory pathways
will also be dysfunctional in parallel disorders of other
tissues or organs. Careful modeling of these different
relationships within the phenome (the set of all phe-
notypes of an organism) or within the interactome
(the set of all interactions of an organism) can greatly
increase the power to identify functional genomic vari-
ation important in disease phenotypes and lead to a
better understanding of how to readjust the system to
return to a normal state.
At its most basic, systems biology comprises three
challenges: (i) how to generate a sufficient quantity
of data to analyze variability in networks, (ii) how to
properly integrate data from multiple disparate
sources into a usable corpus of knowledge, and (iii)
how to use that corpus of knowledge to model a com-
ponent system and optimize it. Model construction
allows predictions of the behavior of a system and, ulti-
mately, an understanding of how to change that behav-
ior in predictable ways. The promise of this approach
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Part II Concepts in Molecular Biology and Genetics
is vast because the exact definition of a system is not
fixed—anything from a particular biochemical path-
way up to the level of an entire organism can be con-
sidered a system in biomedical applications. In this
regard, population-level biosciences, such as epidemi-
ology and population genetics, have long practiced a
form of systems biology, but their focus has always
been on a collection of individuals as the system of inter-
est. Current systems biology approaches model systems
of interest to clinical outcomes, such as metabolic path-
ways, individual cells, and organs. Modeling at this level
has the appeal of allowing translation of results from
decades of population-based laboratory research to be
applied to individualized clinical medicine.
The field of systems biology borrows from control
theory, which deals with the behavior of dynamic sys-
tems (systems that fluctuate in a dependent fash-
ion). Control theory proposes the idea that systems
can best be modeled as a cycle of controllers which
modify inputs to produce the desired outputs, and
sensors which provide feedback to the system, pro-
ducing a steady-state system in which equilibrium is
achieved by balancing the input and output concen-
trations and the feedback signals. In a similar fash-
ion, systems biology includes the definition and
measurement of the components of a system, formu-
lation of a model, and the systematic perturbation
(either genetically or environmentally) of and
remeasurement of the system. The experimentally
observed responses are then compared with those
predicted by the model, and new perturbation
experiments are designed and performed to distin-
guish between multiple or competing models. This
cycle of test-model-retest is repeated until the final
model predicts the reaction of the system under a
broad range of perturbations.
Systems Biology as a Paradigm Shift
Although systems biology is itself a new discipline, the
study of systems in biology is not new. Early studies of
enzyme kinetics employed a similar cycle of testing
and modeling, followed by retesting of new hypoth-
eses. In a similar fashion, modeling of neurophysio-
logical processes like the propagation of action
potentials along a neuron are illustrative of early for-
ays into mathematical modeling of cellular processes.
However, like all next-generation methodological
shifts, systems biology involves a rethinking of basic
principles—a return to a more basic understanding
or a more simplistic framework in which to generate
hypotheses. Traditional science follows a bottom-up
paradigm—that is, break a problem down into its indi-
vidual components (reductionism), learn everything
there is to know about those individual components,
and then integrate the information together to get
information about a system. Systems biology can work
within this paradigm, creating models for individual
components, and then integrating the models (creat-
ing, in essence, models of models, to explain the behav-
ior of the original system). However, this approach
186
requires that knowledge of the individual components
is sufficient to explain the behavior of the system. Apart
from a few exceptions, current reductionist research
models have not been successful at explaining the
large-scale behavior of biologic systems. To this end,
systems biology endorses a more top-down approach,
allowing modeling to occur at a higher level of organi-
zation (not at the level of the components, but at the
level of the organ or the individual). In this point of
view, a better understanding of the system as a whole
can create a better understanding of the components.
“Systems biology . . . is about putting together rather
than taking apart, integration rather than reduction.
It requires that we develop ways of thinking about inte-
gration that are as rigorous as our reductionist pro-
grammes, but different” [2]. Or, put more simply [3]:
“You [can] study each part of a Boeing 777 aircraft
and describe how it functions, but that still wouldn’t
tell you how the airplane flies.” This has led to the
recognition that new paradigms (integration rather
than reduction) may be necessary to understand and
model systems with multiple interacting quantitative
components.
DATA GENERATION
Central to the success of any modeling endeavor is
the generation of large quantities of data. Because
of the increased interest in systems biology, the
theme of the late 1990s and early 21st century
reflected exactly this necessity, exemplified by the
fact that several papers were published on methods
of dealing with the so-called “data deluge” [4–6].
Genomic technologies were the first to enter the
high-throughput realm and subsequently the first
to produce large quantities of high-quality data. Cou-
pled with a concurrent explosion in computing
power, high-throughput data generation made realis-
tic modeling feasible. New technologies produced
during this time vastly increased the ability to query
an entire system at once and led to the advent of
the major international efforts of the early 21st cen-
tury, such as the Human Genome Project (the effort
to sequence the entire complement of human chro-
mosomes) [27]. The HapMap Project (the effort to
catalog common genetic variation across multiple
human ethnic populations) [7], the ENCODE Project
(basically a merger of the Human Genome and Hap-
Map projects—an effort to resequence particular por-
tions of the human genome in multiple individuals
from different ethnic populations to explore com-
mon and rare genetic variation at a higher resolu-
tion) [8], and the 1000 Genomes Project (an effort
to completely sequence 1000 human genomes) [28].
Specifically, microarray technology coupled with
advances in mass spectroscopy, combinatorial chemis-
try, and robotics created an explosion of data and
unique visualizations of cellular processes. Capitaliz-
ing on this explosion of data, the first quantitative
model of the metabolism of a whole (hypothetical)
cell was published in 1997 [9].
 Chapter 10 Integrative Systems Biology: Implications for the Understanding of Human Disease
Microarrays
Microarray technology grew out of a confluence of trends
and technologies. On the experimental level, microar-
rays are most similar to Southern blotting, where frag-
mented DNA is attached to a substrate and then probed
(hybridized) with a known gene or fragment to identify
complementary sequences. The trend through the
1990s was to increase capacity in individual experiments,
allowing the analysis of more and more samples (or more
and more markers) in a single experiment. With the
advent of paper-blotting techniques (allowing DNA
molecules to be immobilized or spotted on special paper,
then hybridized), and subsequently glass-blotting techni-
ques, coupled with advances in robotic pipetting (allow-
ing smaller and smaller volumes of liquid to be spotted,
in greater and greater densities), microarrays became
feasible. Initial microarray experiments [10] began
with small numbers of immobilized probes, owing
mostly to limited knowledge about the genes that make
up a genome. This was quickly followed by arrays
with hundreds, thousands, tens of thousands, and now
(currently) millions of probes as our understanding of
the components of genomes grew (owing, in large part,
to early efforts to identify and catalog all expressed genes
in organisms and to large-scale efforts like the Human
Genome and ENCODE projects).
Microarrays have now found use in multiple experi-
mental venues. Most commonly, the molecules being
immobilized on the array are DNA molecules, allowing
measurement of expression levels or detection of poly-
morphisms (such as single-nucleotide polymorphisms
or SNPs). A DNA microarray consists of thousands of
microscopic spots (or features), each containing a spe-
cific DNA sequence. Each feature is typically labeled
with a fluorescent tag and then used as probes in
hybridization experiments with a DNA or RNA sample
(the target), which is labeled with a complementary
fluorescent label. Hybridization is quantified by fluo-
rescence-based scanning of the array, allowing deter-
mination of the relative abundance of nucleic acid
sequences in the target by characterization of the rela-
tive abundance of each fluorophore (Figure 10.1).
Figure 10.1 Example of an approximately 40,000 probe
spotted oligonucleotide microarray with enlarged inset
to show detail (Image from Wikimedia Commons).
Transcriptomics
Transcriptomics is the study of relative RNA transcript
abundances, using microarray technologies. Chips that
are specialized for this purpose are known as RNA micro-
arrays and are typically prepared with a library of tran-
scripts of known origin (representative tags for a known
complement of genes, for example—the current human
RNA arrays contain approximately 60,000 probes, repre-
sentative of a majority of known RNA species from the
20,000 or so human genes). These are then interrogated
with RNA (typically reverse transcribed into cDNA) from
two different samples, labeled with different dyes (com-
monly green and red dyes). This allows the relative abun-
dance (in one sample versus the other) of each RNA
transcript to be assessed. Experiments of this type are
routinely used to determine which RNAs are upregulated
or downregulated in a disease sample versus a normal
sample. However, note that the utility of the information
generated from transcriptomics studies is highly variable,
as transcription levels are potentially influenced by a wide
range of factors, including disease phenotypes. To be of
general use, transcriptomic data must be generated
under a wide variety of conditions and compared, to
eliminate the trivial sources of variation.
Genotyping
One of the earliest explosions of data on a whole-genome
scale came from early genetic linkage studies. The first
whole-genome genetic linkage scans were performed in
the early 1990s using panels of 350 genetic markers scat-
tered throughout the autosomal chromosomes, as well as
the X-chromosome [11]. Subsequent advances in map-
ping techniques and marker discovery increased the
number of markers in the genetic maps, and included
both sex chromosomes, as well as the mitochondrial
genome. Markers for whole-genome genotyping have fol-
lowed a rather amusing pattern of oscillation between the
use of simple polymorphisms (with two alleles) and com-
plex polymorphisms (with multiple alleles). The earliest
genetic markers to be used (easily observable phenotypes
such as eye color, sex, handedness, and others) were gen-
erally treated as simple binary traits. The first protein bio-
markers (blood group protein polymorphisms and HLA
polymorphisms) were complex, having many alleles with
complicated ethnic and regional variations in frequency.
Restriction fragment length polymorphisms (or RFLPs)
having only two alleles indicating the presence or absence
of a recognition site for restriction enzymes were the next
marker type to be in vogue, and were the first to be sug-
gested as usable for whole-genome analysis because of
their frequency throughout the genome. This was fol-
lowed by the discovery of DNA-length polymorphisms,
in the form of tandem repeated regions (called Variable
Numbers of Tandem Repeats or VNTRs), which are com-
plex polymorphisms. Recognition of the existence of tan-
dem repeats led to the discovery of microsatellite
markers—basically short tandem repeats (STRs), on the
order of 2–5 base pairs repeated several times. The fre-
quency of STRs in the genome enabled generation of
the first whole-genome maps. This was followed in quick
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Part II Concepts in Molecular Biology and Genetics

succession by the discovery of single nucleotide poly-

morphisms (or SNPs), which initially were described as
having only two alleles, and so continue in use as simple
markers (though in fact many SNPs have >2 alleles).
Due to their abundance (estimated at 1 SNP per 100 base
pairs of DNA) and the relative ease of genotyping these
markers, the development of DNA microarray-based gen-
otyping technologies for SNPs occurred quickly. SNPs are
now the current standard for genome-wide genetic stud-
ies. When SNP content on genotyping arrays became suf-
ficiently dense (and also due to findings from other
genomic technologies like array-based comparative gen-
omic hybridization (arrayCGH) techniques), the wide-
spread occurrence of copy number variations (deletions
and duplications) was detected, heralding a return to
more complex (multiallelic) markers again. Current gen-
otyping arrays now contain a mixture of simple polymor-
phic markers (SNPs) and so-called copy-number probes,
allowing the assessment of copy-number changes across
the genome, at a density which provides excellent cover-
age for most ethnic groups (1,000,000 SNPs and nearly
that many copy number probes, meaning each array
carries almost 2 million features). It is expected that chip
densities will continue to grow, allowing for higher-
throughput genotyping. However, at the same time
rapid low-cost sequencing technologies are becoming
available that may well allow for affordable whole-
genome sequence-based assessment of genomic varia-
tion, replacing any need for increased array densities.

Figure 10.2 Omics technologies gather data on numerous

Other Omic Disciplines
Recognizing that many properties of biological systems
are emergent, or a result of complex interactions
between components that are not understandable at
the level of individual system components, scientists
in the 21st century turned to analysis of different levels
of organization in an organism. Naturally, given the
penchant of science for fancy names, each level of
organization had to be given an appropriate name.
For example, since the genome is the entire set of
genes of an organism, genomics is the name given to
the study of the whole set of genes of a biological sys-
tem. Various methods, such as genotyping, sequencing
(examining the linear sequence of DNA) and tran-
scriptomics (examining the expression of all genes in
an organism) can be used to interrogate the genome.
Likewise, given that the proteome is the collection of
proteins expressed in a system, proteomics is the name
given to the study of the proteome. Proteomic methods
include traditional techniques such as mass spectros-
copy—identifying compounds based on the mass-
charge ratio of ionized particles. But just as microarrays
are a modification of traditional genomic techniques
for high-throughput experiments, proteomics modi-
fies the techniques of mass spectroscopy in a similar
fashion—creating experimental and informatics pipe-
lines that allow a sample (like a blood draw) to be par-
titioned into various fractions, have those fractions
examined via mass spectroscopy, and have the results
of the mass spectroscopy experiments compared to
levels. Various “omics” fields are displayed here along with the
laboratory techniques used to generate the data, as well as the
relationship of data from one level to another. Adapted from [25].
other mass spectroscopy profiles to allow for identifica-
tion of individual components.
Other omic disciplines are similarly named—the gen-
eral rule being that the respective discipline arises from
the study of the associated “ome” (or set). This gives rise
to disciplines such as (i) metabolomics—the study of the
entire range of metabolites taking part in a biological pro-
cess; (ii) interactomics—the study of the complete set of
interactions between proteins or between these and other
molecules; (iii) localizomics—the study of the localization
of transcripts, proteins, and other molecules; and (iv) phe-
nomics—the study of the complete set of phenotypes of a
given organism. These various “omes” can be organized
hierarchically, as demonstrated in Figure 10.2, based on
the relationships recognized from years of biological
experimentation. As with genomics and proteomics,
experimental procedures for these other “omics” disci-
plines are adaptations of traditional methods (such as
microscopy for localizomics, or single metabolite measure-
ments for metabolomics) for high-throughput protocols.
DATA INTEGRATION
While data generation is certainly of paramount impor-
tance for systems biology, because the technologies that
generate the data have their own unique (and often

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 Chapter 10 Integrative Systems Biology: Implications for the Understanding of Human Disease
proprietary) formats for storing the data, systems biolo-
gists must also concern themselves with integrating data
that span multiple resources. Since 1998, the number of
recognized online databases related to biological infor-
mation has increased 10-fold (from just under 100 in
1998 to over 1000 in 2008) [12]. Resources like the
Bioinformatics Links Directory (http://bioinformatics.
ca/links_directory/) extend this further by collecting
links to molecular resources and tools as well as data-
bases, and currently list 2300 links. However, the problem
is more than just quantity. Issues of data quality, lack of
standards, lack of interfaces allowing for integration,
and longevity (or lack thereof) continue to plague online
biological data resources, and make cross-resource query-
ing or integration difficult [12,13]. Nonetheless, tools con-
tinue to appear to assist in the integration of data from
disparate sources [14]. Using technology adopted from
the burgeoning Semantic Web (or Web 2.0) projects
[15], biologists and bioinformaticists are able to capture
instances of data sufficient for systems biology scale efforts.
Semantic Web Technologies
Many of the problems inherent to integration of
biological data resources are similar to those being
faced by the larger community of World Wide Web
users. The Semantic Web is a vision for how to have com-
puters infer information relating one web page (or ele-
ment) to another [15]. It is an extension of the
current web protocols (primarily the HyperText Trans-
port Protocol or HTTP), which allows for meaning to
be imbedded together with content, such that auto-
mated agents can make associations between data with-
out needing user input. In essence, creating the
Semantic Web involves recasting the information in
the World Wide Web (which currently stores relation-
ships in the form of hyperlinks, which can link anything
to anything, and so do not represent information con-
tent or meaning) into a format which allows relation-
ships to be represented. The eXtensible Markup
Language (or XML) is an early example of this type of
recasting—allowing content to be stored with represen-
tative tags that describe the content. Resource Descrip-
tion Framework (or RDF) builds on XML by using
triples (subject-predicate-object) to represent the infor-
mation in XML tags (or in hyperlinks), and defining a
standard set (or schema) of RDF triples to describe a
particular object. Each part of a triple names a resource
using either a Uniform Resource Identifier (URI) or
Uniform Resource Locator (URL), or a literal. The
advantage of this format is that RDF schemas are prede-
fined so that meaning can be imbedded in the defini-
tion, or by using a hierarchy or ontology, describing
the relationship within and between schemas. Also,
since the RDF triple can contain a location as well as
attribute-value pair, the components of a schema do
not need to be located in the same place. Thus, if we
have a schema representing a web page in which the sec-
tions (header, footer, left panel, right panel, title, and
others) are defined by RDF triples, these web pages
can easily integrate data from multiple sources.
The representation of information by RDF allows the
location of data and data resources to be independent
of the location of user interfaces or analytic resources.
In essence, this allows the integration of data from multi-
ple repositories and the querying of the integrated data.
Coupled with the concept of RDF schemas to accurately
describe knowledge about objects and ontologies to orga-
nize the relationship of objects to one another, the use of
RDF can solve several of the problems mentioned for
data integration (namely, the lack of standards and the
lack of interfaces for integration). Another current
trend—the use of wikis (a website that allows collabora-
tive editing of its content by its users)—addresses the
problem of data quality by allowing users to mark data
as reliable or not, or by allowing users to update out-of-
date or incorrect data.
MODELING SYSTEMS
Once the appropriate types of data have been generated,
and the various sources of data collected and integrated,
the resulting information is turned into knowledge by
interpreting what the data actually mean, and how they
address questions that need to be answered. Data model-
ing is used to understand the relationships important in
defining the system. As noted previously, systems biology
draws heavily from control theory, which itself is derived
from mathematical modeling of physical systems.
Although the mathematical derivation of control prob-
lems is complex, the fundamental concepts governing
the formulation of control models are more intuitive
and relatively limited. Three basic concepts can be
thought of as central to forming control models: (i) the
need for control (regulation or feedback), (ii) the need
for fluctuation, and (iii) the need for optimization. A sim-
ple control model is presented in Figure 10.3.
Key characteristics of this model include a control-
ler (responsible for the conversion between input
and output) and sensor (responsible for determining
the degree and direction of the feedback)—each of
which can be the result of a single or multiple ele-
ments. An initial model of this form can often be
derived from an initial data generation step, which
can measure a baseline set of conditions for the system
and also provide an idea of the components of the sys-
tem. For example, an expression network (groups of
genes that are co-regulated in some fashion) that regu-
lates a biochemical pathway can be thought of as a
control model. A single transcriptomics experiment
can give you information on genes that are potentially
co-regulated (sets of genes that are overexpressed
compared to control, and so which might be providing
the function of a controller), as well as information
about potential sensors (genes that differ in re-
sponse—one being overexpressed, the other under-
expressed). However, the problem with a single exper-
iment (or a single snapshot of the transcriptome) is
that the information derived is not sufficient to disen-
tangle the true positives (elements that truly should be
components of the model) from the false positives
(random variation which transiently mimics the behavior
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Part II Concepts in Molecular Biology and Genetics

Figure 10.3 Example of a control module. This module represents a simple feedback loop, with output from the sensor
either upregulating or downregulating the process that converts the input into output.

Figure 10.4 The iterative nature of systems biology research. Note that every refinement of the model needs additional
data generation for retesting of specific hypotheses. Adopted from [26].

of the model). This reflects the need for fluctuation. By
perturbing (changing an aspect of the system to produce
a predictable outcome) and remeasuring the system,
additional confidence regarding the true positives can
be achieved assuming the new measurements reflect
the predictions of the model. If not, another model
which explains the previous data measurements is
selected, and another test is devised. This cycle continues
until the final model accurately represents the data
measured in all tests. In this way, the most optimized
model is selected (Figure 10.4).
Lastly, although not immediately obvious from the
preceding discussions, data modeling also requires large
amounts of computational power. Due to the size of the
high-throughput data sets currently in use (expression
data on 30,000 genes for multiple time points; SNP geno-
types for millions of markers; occurrence of all known
protein-protein interactions; computational prediction
and annotation of all known protein-DNA binding sites;
and others), the advanced mathematical and visualiza-
tion frameworks currently in use, and the iterative nature
of analysis, large amounts of computing power are nec-
essary, often pushing the limits of even parallelized or
grid-enabled computational clusters. Continued growth
in computing power will be necessary to support the
ongoing use of systems biology as the models in use
become more and more elaborate and incorporate
information from greater numbers of high-throughput
methodologies.
IMPLICATIONS FOR UNDERSTANDING
DISEASE
A new revolution in medicine was made possible at the
end of the 20th century by the sequencing of the human
genome. This remarkable feat led to the revelation that
millions of variations exist in the DNA sequence of indi-
vidual humans, with an infinite number of possible var-
iations. Furthermore, genetic studies revealed that
many chronic inflammatory diseases, such as Crohn’s
disease, ulcerative colitis, chronic pancreatitis, rheuma-
tologic disease, heart diseases, and others do not have a
single genetic factor causing the disease, but rather
several dozen common genetic variations that, in the
context of many possible interacting environmental fac-
tors, cause a disease that is defined by the location of
inflammation and associated signs and symptoms.
Unfortunately, the simplicity and early success of the
germ theory of disease, in which a single pathological
agent was responsible for a specific disease with charac-
teristic signs and symptoms, lulled physicians into think-
ing that all disease would follow a similar pattern. This
reductionist-like approach in medicine allowed physi-
cians and scientists from different disciplines to triangu-
late on the same target using different methods and
perspectives. It has identified numerous components
of individual systems—many of which are reused over
and over, leading to a modular view of system compo-
nents—and has provided numerous insights into

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 Chapter 10 Integrative Systems Biology: Implications for the Understanding of Human Disease
human disease. Despite these advances, the reductionist
approach in medicine has been less successful in identi-
fying the many complex interactions between disease
components, and in explaining how system properties
(like disease phenotypes) emerge—a concept which
becomes important when considering manipulation of
systems to produce predictable and desirable outcomes,
as required for preventative medicine. As such, new
paradigms are required, likewise requiring new
approaches and new methods.
One such new paradigm is the advent of persona-
lized medicine, which can be thought of as an applica-
tion of systems biology-type thinking to medicine.
Personalized medicine represents a transition from
population-based thinking (describing risks on a popu-
lation level) to an individual-based approach (describ-
ing risks for an individual based on his or her personal
genomic/proteomic/metabolomic/phenomic profiles).
Reductionist approaches have successfully identified
many of the biomarkers required to define disease states
in complex disorders on a population level (that is
attributing population risks to various changes), but have
not been able to describe how individual exposures
or biomarkers (or combinations of these components)
interact to create the disease state in individuals. To
achieve this transition, systemic models which explain
the function of systems (cells, organs, etc.) are needed,
from which the implications of changes in particular
exposures or biomarkers (genetic changes, proteomic
changes, and others) can be understood on the system-
level.
Redefining Human Diseases
The first major hurdle in transitioning from allopathic
medicine to personalized medicine is to redefine com-
mon human diseases. In allopathic medicine, diseases
are typically defined by characteristic signs and symp-
toms that occur together and meet accepted criteria.
Chronic inflammatory diseases are defined by the loca-
tion of the inflammation, the persistence of inflamma-
tion, and the presence of tissue destruction or
scarring. These definitions indicate that the specific
mechanism causing a specific organ to develop and
to sustain an inflammatory reaction is not known. Fur-
thermore, it has been impossible to identify the cause
when using the traditional scientific methods used to
identifying a single, causative infectious agent. Instead,
the possibility that disease affecting one or more
organs can occur through any one of multiple path-
ways, and that each pathway has multiple steps and
regulatory components, and that multiple effects on
multiple systems and multiple environmental expo-
sures may be required before disease is manifest must
be embraced to transition to personalized medicine.
Indeed, physicians recognized that diseases located in
specific organs share some common systemic features
such as the type of inflammatory response (autoim-
mune or fibrosing), or chronic pain since they use a
limited number of anti-inflammatory or pain medica-
tions for a variety of diseases. Additionally, studies
which have looked at the clustering of disease phe-
notypes based on their representative genomics
(associated gene/SNP polymorphisms or expression
profiles) [16] have demonstrated that even disease phe-
notypes we once thought were roughly homogeneous
(for instance, disease subtypes like Crohn’s disease with
ileal involvement in Caucasian-only populations) are in
fact associated with different genetic loci (heteroge-
neous) [17]. Personalized medicine must deconvolute
systemic and tissue-specific pathways and reconstruct
them in a way that leads to precise, patient-specific
treatments.
The Transition to Personalized Medicine
The effect of approaching complex inflammatory dis-
orders as a single disease rather than as a complex pro-
cess is illustrated through the comparison of multiple
small studies by meta-analysis. While some of the
variance in estimated effect sizes from small genetic
studies can be attributed to random chance, the pos-
sibility also exists that populations from which the
samples were taken were not equivalent, and that dif-
ferent etiologies will lead to the same end-stage signs
and symptoms through different, parallel pathways.
In cases where a candidate gene is critical to some
pathologic pathway, but not others, the wide variance
between small genetic studies may reflect the fraction
of subjects that progress to disease through that spe-
cific gene-associated pathway.
Evidence of this effect was recently demonstrated in
an evaluation of reports on the effect of the pancreatic
secretory trypsin inhibitor gene (SPINK1) N34S poly-
morphism in chronic pancreatitis [18]. A model was
developed to test the hypothesis that alcohol, which
is known to be associated with chronic pancreatitis
and fibrosis via the collagen-producing pancreatic stel-
late cell (PSC), drives pancreatic fibrosis through a
recurrent trypsin activation pathway as seen in heredi-
tary pancreatitis [19,20], or through a trypsin-indepen-
dent pathway, as illustrated in Figure 10.5 [18].
We identified 24 separate genetic association stud-
ies of the effect of the SPINK N34S mutation on
chronic pancreatitis with effect sizes (odds ratio, OR)
reported to be between nonsignificant to 80. Using
meta-analysis, we determined an overall effect of the
SPINK1 pN34S on risk of chronic pancreatitis to be
high (OR 11.00; 95% CI: 7.59–15.93), but with signifi-
cant heterogeneity. Subdividing the patients into four
groups based in proximal etiological factors (alcohol,
tropical region, family history, and idiopathic), we
found that the effect of SPINK1 pN34S on alcohol
(OR 4.98, 95% CI: 3.16–7.85) was significantly smaller
than idiopathic chronic pancreatitis (OR 14.97, 95%
CI: 9.09–24.67) or tropical chronic pancreatitis (OR
19.15, 95% CI: 8.83–41.56). Thus, we conclude that
alcohol acts through Factor A-type pathway, while trop-
ical chronic pancreatitis and idiopathic chronic pan-
creatitis act through trypsin-associated pathways. The
fact that a higher percentage of alcoholic patients than
the control populations had SPINK1 mutations may
191
Part II Concepts in Molecular Biology and Genetics
Figure 10.5 Hypothesis of etiology-defined pathways to pancreatic fibrosis. Hypothetical influence diagram illustrating
pathologic pathways linking proximal factor (Factor A and B) to PSC (pancreatic stellate cell) and fibrosis through multiple
steps (a1, a2, a3). Etiological factors of type B activate trypsinogen to trypsin, and therefore their pathologic pathway to the
PSC can be interrupted by SPINK1. Etiological factors of type A are independent of trypsin, and therefore will not be
influenced by variations in SPINK1 expression or function.
also mean that some idiopathic patients were misdiag-
nosed as being alcoholics since the threshold for
alcohol-associated risk was previously unknown [21].
It could also mean that alcohol accelerates the pro-
gression from recurrent acute pancreatitis to chronic
pancreatitis as demonstrated in animal models [22].
Thus, these results are important for understanding
the etiology and progression of alcoholic chronic
pancreatitis, but have broader implications for under-
standing the presence and effects of heterogeneity of
pathways in complex disorders.
The chronic pancreatitis model also illustrates a
major problem for transitioning to personalized medi-
cine. Very large association studies require the recruit-
ment of subjects from many large medical centers
in different regions and different countries. This
approach will tend to obscure mechanism-based het-
erogeneity and converge on only the most common
features of the disease (population-level effects), even
though other factors may have a stronger biological
effect in a limited number of patients, while being
irrelevant in others. This argues for more detailed phe-
notyping of study populations (phenomics) and careful
analysis of context-dependent effects (interactomics).
The application of systems biology to this data would
allow for a better understanding of the various pathways
leading to disease states, and so a better understanding
of the possible interactions (or context-dependent
effects) that accurately predict disease in a single
individual.
Applications of Systems Biology to Medicine
The realization of personalized medicine requires not
only a more systems-like perspective regarding risk
factors, it also requires a rethinking of existing classifi-
cations, which are largely derived from observation
and reductionist approach (common observable phe-
notypes should be the result of common underlying fac-
tors). As an example of this rethinking, a recent study
undertook the reclassification of the disease phenome
(the space of all associations between disease pheno-
types) using the explosion of current information on
genetic disease associations [23,24]. Disease-associated
genes were clustered based on information about gene-
gene or protein-protein interactions (from transcrip-
tomics or interactomics studies), and superimposed with
a representation of the organ system of the associated
192
disease phenotype. The resulting network graph (Figure
10.6) demonstrates the association of different genes
together in modules, many of which represent asso-
ciations of proteins in macromolecular complexes
(protein-protein interactions), or association of proteins
in metabolic or regulatory pathways.
This view of the network of disease genes can at the
same time inform us about novel associations we might
not have been aware of (such as PAX6 in the ophthal-
mological cluster, or PTEN and KIT in the cancer clus-
ter), and also direct us toward pathways (clusters) that
might be of most benefit in understanding the net-
work more completely (that is, those that are involved
in multiple disease states or that have the most connec-
tions to them). This network-centric view of disease
can also inform clinical medicine in terms of the
choice of medications (indicating that medications
used in one disease might also function appropriately
in another, based on clustering of associated genetic
factors).
DISCUSSION
We have outlined many of the reasons that a systems
biology-based approach is needed for understanding
complex disorders. Once the key components are
organized into a logical series, then formal modeling
of the disease process can be applied, tested, addi-
tional experimental data added, the system calibrated
and retested. The optimal model or models are yet to
be determined experimentally for any system, but
with the continued rapid increase in high-throughput
data generation and the continued increase in
computational power, large-scale integrations and
models can be achieved. The challenge will be to
accurately anticipate the information necessary to be
included in the model, as accurate assessment of the
context is essential to the appropriate understanding
of the effect of individual variation and the integra-
tion of that understanding into clinical practice. It is
not enough to know that certain changes affect a phe-
notype (like disease risk) in a population, as these
overall effects are typically very small (on the order
of an odds ratio of 1.1 to 1.2). However, with a proper
understanding of the context in which each variant is
important, appropriate medical interventions can be
implemented.
 Chapter 10 Integrative Systems Biology: Implications for the Understanding of Human Disease
Figure 10.6 Disease gene network. Each node is a single gene, and any two genes are connected if implicated in the same
disorder. In this network map, the size of each node is proportional to the number of specific disorders in which the gene is
implicated. Reproduced with permission from [24].
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