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Drug Discov Today. 2009 August ; 14(15-16): 754–760. doi:10.1016/j.drudis.2009.05.005.

New tools for functional genomic analysis

Xin Chen1, Eric Jorgenson1,2,3, and Siu Tim Cheung4

1Dept. of Bioengineering and Therapeutic Sciences University of California, San Francisco, CA

2Dept. of Neurology, University of California, San Francisco, CA

3Gallo Clinic and Research Center, Emeryville, CA
4Dept. of Surgery, The University of Hong Kong, Hong Kong, China

Abstract
For the past decade, the development of genomic technology has revolutionized modern biological
research and drug discovery. Functional genomic analyses enable biologists to perform analysis of
genetic events on a global scale and they have been widely used in gene discovery, biomarker
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determination, disease classification, and drug target identification. In this article, we provide an
overview of the current and emerging tools involved in genomic studies, including expression arrays,
microRNA arrays, array CGH, ChIP-on-chip, methylation arrays, mutation analysis, genome wide-
association studies, proteomic analysis, integrated functional genomic analysis and related
bioinformatic and biostatistical analyses. Using human liver cancer as an example, we provide further
information of how these genomic approaches can be applied in cancer research.

Keywords
Functional genomics; arrays; cancer

Genomic analyses include a variety of tools that address the global changes of specific
biological parameters. Genomic analyses that examine DNA, RNA, or protein levels provide
powerful tools to characterize gene function and regulation, facilitate disease classification,
biomarker identification, risk factor stratification and drug discovery (Fig. 1).

Global Gene Expression Profile

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Genome-wide expression studies empowered by microarray analysis enable the systematic

analysis of complex biological systems and because expression microarrays were developed
in the 1990s they represent the beginning of the genomic era. The principle of microarray
expression profiling study is based on the hybridization of a single-strand nucleic acid fragment
to its complementary single strand with high specificity [1]. The target cDNA is first labeled
using fluorescent dyes and then hybridized to the array surface. The retained labeled target will
then be subjected to stringent washes, capture and quantification of the fluorescent signals,

© 2009 Elsevier Ltd. All rights reserved.

Corresponding author: Chen, X (E-mail: E-mail: [email protected] Tel: 415-502-6526; Fax: 415-502-4322) or Cheung, ST (Email: E-
mail: [email protected] Tel: 852 2819 9651; Fax: 852 2819 9634).
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This article provides an overview of the current and emerging tools involved in genomic studies and analyses.
 Chen et al. Page 2

followed by the data analysis process. Two platforms that are commonly in use are: cDNA
microarrays and oligonucleotide microarrays [1]. The cDNA microarrays contain a collection
of probes generated by PCR amplification from cDNA libraries, expressed sequence tag clones
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or long genome cloned fragments. Oligonucleotide microarrays commonly contain short

oligonucleotides (25–30 nt) or long oligonucleotides (50–80 nt). In both platforms, the short
DNA segments are printed onto solid supports, usually microscopic slides, by direct contact
mode (mechanical robotic spotting) or non-contact mode (ink jet technique). Oligonucleotide
microarrays manufactured by Affymetrix, the GeneChip, employ a different technology
platform, designated the photolithographic method, to synthesize the short oligonucleotides
in situ on the chip. Different platforms each have their limitations and advantages, but in
common, all have been shown reliably to capture gene expression signature on a genomic scale
[1].

Global expression profiles enable a better understanding of the molecular signature of human
diseases, including liver cancers [2,3] (Fig. 2). For instance, we and others have reported
genome-wide expression profiles of liver cancer and their clinico-pathological implications
[4–7]. We have observed specific gene expression patterns between tumor and non-tumor
[4], in association with p53 status [4], and clonality delineation for multi-nodular tumor [8].
There are reports on gene expression profiling in association with tumor metastasis [6], and
patient outcome [5,7]. Differentially expressed genes demonstrated the potential to serve as
prognostic biomarkers [9] and therapeutic targets [10].
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Genomic Analysis Small non-coding RNAs

An emerging number of studies have shown the regulation of gene expression by small non-
coding RNAs in animals and plants [11,12]. MicroRNAs (miRNA) are an abundant class of
small non-protein-coding RNAs that are 19- to 25-nucleotides in length that have been
implicated in many human diseases including tumor initiation and progression. miRNA
function as negative gene regulators, which can control hundreds of gene targets, and may
function as either tumor suppressors or oncogenes [13]. Differential expression of miRNAs
have revealed diagnostic, prognostic and therapeutic implications [14]. There are several
approaches for genome scale miRNA expression profiling [15,16]. One of the most commonly
used platforms is miRNA oligo arrays [17]. The overall experimental design of these miRNA
arrays is very similar to regular expression arrays. Other miRNA profiling methods included
multiplexed q-RT-PCR assays and bead based methods [15,16].

Array based CGH for Genome-wide Analysis of DNA Copy Number Variation
Chromosomal imbalances, including deletions and amplifications, are common in human
tumors. Comparative genome hybridization (CGH) has been widely used to examine for the
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global analysis of DNA copy number since its first report in the early nineties. The resolution
of conventional CGH is limited by the length of the metaphase chromosomes, which is
approximately 10 megabases and could contain hundreds of genes. Microarray-based CGH
has been developed, which combines microarray technology with the CGH approach [18,19].
Defined DNA fragments (BACs, cDNAs or oligo) have been used to replace metaphase
chromosomes and results in higher resolution. Microarray-based CGH allows a precise
mapping for the regions of genetic aberrations [20,21], including human liver cancers [22].

Genome-wide Analysis of DNA Methylation

DNA methylation is one of the most important covalent modifications of genomic DNA in
eukaryotic cells. Human tumor samples frequently show abnormal patterns of DNA
methylation, which may contribute to the aberrant patterns of gene expression and regulation.
Traditional, methylation can only be determined on a gene-by-gene basis using methods that

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include bisulfite conversion followed by PCR or sequencing, methylation-sensitive restriction

enzymes or affinity purification. Recent technology development has enabled the analysis of
DNA methylation in a genome-wide scale [23,24]. Genome-wide methylation analyses can be
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divided into two categories: array based or non-array based. Several companies provide
commercially-available arrays for methylation analysis [24]. The arrays are designed to
analyze bisulfite-converted DNA (for example, bead arrays from Illumina), or use the
restriction enzyme-based methylation analysis (for example, oligo arrays from NimbleGen or
Agilent). Array hybridization and analysis is similar to what has been described in expression
or CGH arrays. Nonmicroarray based experimental design include Restriction Landmark
Genome Scanning (RLGS), methylation specific digital karyotyping (MSDK), and high-
throughput sequencing after bisulfate conversion [23,24].

Chromatin Immunoprecipitation Followed by Microarray Analysis (ChIP-

chip)
“ChIP-chip” applies antibodies specific to a regulatory factor, in most cases, a transcriptional
factor, for genome-scale chromatin-immunoprecipitation combined with microarrays spotted
with intergenic sequences to identify their bound targets [25,26]. Protein-bound DNA
fragments retrieved by the antibody are hybridized to microarrays to identify the retrieved
sequences. ChIP-chip analysis is now being widely used as a reliable tool to identify targets
of critical transcriptional regulators in a high throughput manner.
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Genome-wide Association Studies of DNA Sequence Variation

DNA sequence variation can influence disease risk and response to drug therapy by altering
gene expression, RNA processing, or the amino acid sequence of proteins. Genome-wide
association studies use DNA microarrays to investigate the effect of millions of common DNA
sequence variants in the human genome, of which the most common type are single nucleotide
polymorphisms (SNPs). The genome-wide association approach is statistically powerful [27]
and has led to the discovery of many new genetic variants that underlie variation in human
traits (www.genome.gov/26525384), including a number of cancers [28]. A number of these
disease variants influence the risk of multiple diseases, including shared risk variants for several
common cancers [29]. Thus, the genome-wide studies may have important implications in drug
development by assisting to identify novel therapeutic targets and genetic biomarkers that for
drug discovery [30].

Genome-wide association studies have become possible due to several recent technological
advances. Improvements in DNA microarray technology have rapidly reduced the cost of
genotyping SNPs, allowing for the testing of up to one million SNPs using a single microarray.
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At the same time, the HapMap Project validated nearly four million SNPs in multiple diverse
populations, and determined the extent of linkage disequilibrium (LD) between SNPs [31]. LD
refers to the non-random association of SNPs, typically those that are closest together. The
presence of LD allows for SNPs on genotyping platforms to serve as a proxy for other nearby
SNPs [32]. As a result, current DNA microarrays can assay most common SNPs in the HapMap.
In this way, LD reduces both the genotyping costs of genome-wide association studies and the
multiple testing burden (see the Biostatistical Analysis section below).

It is important to note that genome-wide association studies are better suited to investigate the
potential association of common variants, typically defined as those with a minor allele
frequency of greater than 5%, with disease than rare variants [33]. Since strongly deleterious
alleles are likely to face selection pressure, variants with large effects will be rare; common
variants will have more modest effects on gene function. Most variants associated with disease
in genome-wide association studies are common and have modest or small effects. Because of

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this, individual variants will not serve as strong predictors of disease risk [34]. They may,
however, explain a large amount of the risk of disease in the population, or population
attributable fraction. For this reason, interventions that developed to counteract these risk
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variants could substantially reduce the incidence of disease in a population.

Genome-wide Somatic Mutation Analysis

Human cancer is widely considered to be induced by somatic alterations within the cancer
genome, leading to mutations of oncogenes or tumor suppressors [35]. Sequencing of tumor
cell genomic DNA, also known as “deep sequencing”, has been applied to identify “driver
mutations”, which will clearly have an important impact on our understanding of
carcinogenesis. The development of next-generation sequencing technologies has enabled us
to perform genome-wide mutation analysis on cancer cells [36–38]. In most cases, these deep
sequencing analyses involve amplifying all individual exons for most known genes, followed
by large scale sequencing to examine the amplicon for possible mutations [36,38]. For instance,
recent studies have discovered somatic mutations that affect key signaling pathways in acute
myeloid leukemia and lung cancers [39,40].

Proteomics Analysis
Expression profiling (mRNA-based for expression level changes) and genomic profiling
(DNA-based for copy number or sequence variation) can not provide a complete picture on
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the heterogeneity of complex diseased tissue. The level of mRNA or DNA changes do not
always correspond to protein level changes, nor to the post-translational modifications, e.g.
phosphorylation, which are critical in regulating protein activity. Indeed, a number of the
targeted therapeutic agents are designed to inhibit the activity of a protein, e.g., tyrosine kinases.
Therefore, protein profiling is essential in providing the protein molecular signatures for
bioassay and therapeutic development. Two-dimensional polyacrylamide gel electrophoresis
(2D–PAGE) approach is commonly employed to study protein profiles [41,42]. The proteins
can be separated according to their size (molecular mass) and charge (isoelectric point)
properties and their abundance then determined accordingly. The difficulty in elucidating the
identity of the protein spots remains, however, the major obstacle in clinical application. Since
the late 1980s, matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS)
has been advanced to allow the rapid measurement for the molecular weights of different
proteins with a time-of-flight (TOF) MS. MALDI-TOF MS has limitations on mass resolution
and accuracy, however, to identify peptides with high confidence. Alternative approaches have
been used to provide a more reliable determination of peptide sequence, including collision-
induced dissociation (CID) with a tandem MS, electron capture dissociation (ECD), infrared
multiphoton dissociation (IRMPD), and electron transfer dissociation (ETD) [43].
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Furthermore, protein arrays, also known as antibody arrays, are an emerging technology that
provides parallel analysis of multiple proteins [44]. In addition, protein arrays can be applied
to profile specific protein post-translational modifications, such as phosphorylation or
neddylation, and to measure enzyme activities and protein cell-surface expression. Proteomic
approaches have been widely used for biomarker discovery in human tumors [45]. Protein
profiling of blood samples has been the focus in recent years, because it allows repeated
measurements (especially important in monitoring treatment response) and without the need
to obtain tumor tissues. Blood samples, prepared as serum or plasma fractions, have been used
for biomarker discovery [46–48].

Biostatistical Analysis of Genomic Studies

Most genomic experiments produce large amounts of data. For example, in a gene expression
microarray study, 22,000 genes x 100 samples will generate 2.2 million data points. In addition,
genomic experiments are often noisy and not normally distributed, and usually contain missing

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values in the expression matrix. Robust biostatistical analyses are required to obtain biological
relevant interpretations of the genomic data [49,50].
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Specific statistical tools need to be applied to specific genomic studies. Therefore, people have
to choose biostatistical software that is best suited for their specific experiments and those
questions that they are trying to address. In general, statistical analyses of genomic data can
be divided into two major categories: supervised and unsupervised methods [49,50].
Supervised approaches try to identify the genetic events that fit a predetermined pattern. For
example, supervised analysis is used to identify genes that are differentially expressed between
groups of samples, as well as to find genes that can be used to accurately predict the
characteristics of groups. In contrast to the supervised method, the unsupervised approaches
characterize genomic data without prior input or knowledge of predetermined pattern.
Unsupervised analysis is used to identify internal structure in the genomic data set. The most
commonly used unsupervised analysis tool is Hierarchical clustering and Principal
Components Analysis (PCA).

Because genomic studies examine thousands or millions of data points, stringent significance
criteria are applied to the association results. One method is to undertake a Bonferroni
correction by dividing the significance criteria by the number of tests being conducted. For
example, correcting for a million tested common SNPs in a genome-wide association study,
an association would need a p-value of 5 × 10−8 (0.05 divided by 1,000,000) to be considered
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“genome-wide significant.” It is for this reason that genome-wide association studies typically
involve thousands of subjects to achieve sufficient statistical power. Other multiple testing
adjustments that are less conservative than a Bonferroni correction, such as permutation
derived p-values and false discovery rates, are often employed to maintain statistical power
and to clarify the strength of a reported finding in light of the genomic scale of the experiment
[51,52].

Despite all these advantages of biostatical analysis, there is no standard or one-size-fits-all

solution for statistical approach or even a single way to pick the significant p-value to balance
type I and type II errors in statistical readouts. Each biologist has to approach this question
based on his/her own biological questions in each specific setting.

Bioinformatics Analysis
Genomic studies generally generate large amounts of data. Even after statistical analysis, one
may identify large number of de-regulated genes, for example, genes which are methylated or
mutated in tumor samples. Bioinformatics analysis tools have been developed to assist
scientists to extract meaningful data and interpret the genomic data in a functional manner.
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One of the most commonly used methods to annotate the gene function is through Gene
Ontology (GO, http://www.geneontology.org/) [53,54]. GO classifies gene function according
to three organizing principles: molecular function, biological process and cellular component.
When certain GO terms are statistically enriched in a cluster, it may suggest possible functional
significance of the cluster of genes.

Another commonly used bioinformatic analysis tool is Gene Module Analysis [55,56]. Just as
GO term analysis builds on pre-existing knowledge for the interpretation of microarray data,
one can interrogate the global gene expression profile with respect to known sets of genes by
gene module analysis. In brief, gene module analysis asks whether the genes whose level of
expression changes in an experiment are similar to those which have been observed in another
setting. The gene modules may be defined by function (e.g. GO terms or other annotations),
the presence of specific cis- or trans-regulatory motifs for transcription factor or miRNA
binding, or known responsiveness to specific signaling pathways or drugs. Gene Set

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Enrichment Analysis (GSEA) is the most popular modular analysis method that is publically
available (http://www.broad.mit.edu/gsea/) [57]. Ingeniuty Pathway analysis software
(http://www.ingenuity.com/) is a popular commercially available modular analysis tool.
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Integrated Functional Genomic Analysis

Integrated functional genomics is an approach that combines results from multiple genomic
analyses or genomic analysis and functional experiments to identify important genetic signals
underlying biological processes. Integrated functional genomics is critical for cancer research.
As one can imagine, genome-wide studies are likely to produce large numbers of genes with
expression alternations, mutations, abnormal methylation or DNA copy number variations in
cancer cells. Only a small number of these genetic alternations, however, have functional roles
during tumorigenesis. The majority of the genes are likely to be passenger genes that are either
the products of genomic instability or secondary changes during carcinogenesis, but have no
direct contribution to the malignant transformation. By combining multiple genomic analyses,
one can significantly narrow down the list of genes which may contain functionally significant
oncogenes or tumor suppressor genes. For example, DNA copy number gains and losses
contribute to cancer development by increased and decreased expression of oncogenes and
tumor suppressor genes, respectively. In our recent studies, we combined expression arrays
and array CGH studies of human HCC samples, and were able to identify a relatively small
set of genes as candidate oncogenes or tumor suppressors whose expression levels are
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associated with DNA copy number changes [58]. In another example, ChIP-chip experiments
can be integrated with gene expression analysis to delineate how specific transcriptional factors
regulate global gene expression. In a recent study, Acevedo et al. performed ChIP-chip
experiments to assay binding of RNA polymerase II, H3me3K27, and H3me3K9 and DNA
methylation in 25,000 promoter regions in normal liver and liver tumor samples [59]. The
experiments successfully identified changes in active and silenced regions of the genome in
liver tumor cells, and in so doing identified novel molecular mechanisms that mediate tumor
specific changes in gene expression in the liver. In addition, by combining genomic analysis
and functional screenings, such as siRNA mediated gene silencing, we can rapidly identify
potential driver genetic events. For example, in a recent study, Zender L et al. identified small
regions of recurrent deletions in human liver cancer by genomic analyses [60]. Using
microRNA based short-hairpin RNA libraries, targeting genes within these deleted regions,
the group conducted in vivo RNAi screening to identify genes that, when silenced, cooperate
with Myc to promote liver cancer development. The study successfully identified and validated
13 tumor suppressor genes for liver cancer [60].

Conclusion
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In summary, in the next decade, with these tools for genomic analyses being widely used in
biomedical research, one can foresee the emerging of large amounts of data tangling different
biological questions. Functional genomic analyses will likely have multiple implications for
drug discovery and development. For example, integrated functional genomic studies will
likely identify driver mutations or genes which tumor cells depend on for growth and
metastasis. These genes can be used as targets for drug development, and it will lead to drugs
for specific genetic events which are likely to be more efficient and less toxic for cancer
treatment. Genomic analyses will also identify genetic signatures, such as gene expression
profiles or specific mutation status, which can be used to predict drug responsiveness. These
biomarkers will clearly increase the power and efficiency of clinical trials by selecting the
appropriate patient populations and may lead to successful clinical drug development.
Altogether, the application of genomic analysis to drug development will facilitate drug
discovery and the development process in a more efficient manner.

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ACKNOWLEDGEMENT
This work is supported by NIH K01CA096774 and R21CA131625 to X.C as well as RGC and NSFC (HKU 7560/06M
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and N_HKU 709/07) to S.T.C.

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Figure 1.
Schematic illustration of new tools for functional genomic analysis

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Figure 2.
Functional genomic approach in liver cancer studies using gene expression profiling. Liver
tissues and liver cancer tissues were collected with patient consent. The purified nucleic acid
samples would be labeled with fluorescent dyes and hybridized to the arrays. The fluorescent
signal intensities would be further analyzed by biostatistic and bioinformatic methods [4]. The
clinical implication for functional genomics includes expression finger printing to delineate
the clonality relationship of the multinodular tumors [8], identify novel biomarkers for
prognostication [9] and therapeutic development [10].

Drug Discov Today. Author manuscript; available in PMC 2010 August 1.