OMICS A Journal of Integrative Biology

Volume 18, Number 11, 2014

Mary Ann Liebert, Inc.
DOI: 10.1089/omi.2014.0062

Novel Multivariate Methods for Integration

of Genomics and Proteomics Data:
Applications in a Kidney Transplant Rejection Study
Oliver P. Gunther,1,2 Heesun Shin,1,7 Raymond T. Ng,1,8 W. Robert McMaster,1,4,9
Bruce M. McManus,1,3,7,10 Paul A. Keown,1,3,5,6 Scott. J. Tebbutt,1,7,10,11 and Kim-Anh Le Cao12,13

Abstract

Multi-omics research is a key ingredient of data-intensive life sciences research, permitting measurement of
biological molecules at different functional levels in the same individual. For a complete picture at the biological
systems level, appropriate statistical techniques must however be developed to integrate different omics data sets
(e.g., genomics and proteomics). We report here multivariate projection-based analyses approaches to genomics
and proteomics data sets, using the case study of and applications to observations in kidney transplant patients
who experienced an acute rejection event (n = 20) versus non-rejecting controls (n = 20). In this data sets, we show
how these novel methodologies might serve as promising tools for dimension reduction and selection of relevant
features for different analytical frameworks. Unsupervised analyses highlighted the importance of post transplant
time-of-rejection, while supervised analyses identified gene and protein signatures that together predicted rejection status with little time effect. The selected genes are part of biological pathways that are representative of
immune responses. Gene enrichment profiles revealed increases in innate immune responses and neutrophil
activities and a depletion of T lymphocyte related processes in rejection samples as compared to controls. In all,
this article offers candidate biomarkers for future detection and monitoring of acute kidney transplant rejection, as
well as ways forward for methodological advances to better harness multi-omics data sets.
Introduction

ulti-omics research has been at the epicenter of

the post-genomics research agenda, raising both promises and challenges, to extract the best value out of such
data-intensive form of life sciences inquiry (Altman, 2013;
Gomez-Cabrero et al., 2014), not to mention the pressing
need for novel methods for analysis of multi-omics data sets
(Gomez-Cabrero et al., 2014).
Indeed, recent advances in high throughput omics
technologies now enable quantitative measurements of
expression or abundance of biological molecules of a whole
biological system and at different cellular levels. The improvement of analytical techniques to quantify different

levels of gene products (mRNA, proteins, metabolites)

permits understanding of cell metabolism as one integrated system rather than as a combination of different
parts (Zhang et al., 2010). Whilst single omics analyses are
commonly performed to detect between-groups difference
from either static or dynamic experiments, the integration
or combination of multi-layer information is required to
more fully unravel the complexities of a biological system.
Data integration relies on the currently accepted biological assumption that each functional level is related to each
other. Therefore, considering all the biological entities
(genes, proteins, metabolites) as part of a whole biological system is crucial to unravel the complexity of living
organisms.

NCE CECR Prevention of Organ Failure (PROOF) Centre of Excellence, Vancouver, British Columbia, Canada.
Gunther Analytics, Vancouver, British Columbia, Canada.
3
Department of Pathology and Laboratory Medicine, 6Medicine, 8Computer Science, 9Medical Genetics, 7James Hogg Research Centre,
St. Pauls Hospital, 11Department of Medicine, Division of Respiratory Medicine, University of British Columbia, Vancouver, British
Columbia, Canada.
4
Immunity and Infection Research Centre, Vancouver, British Columbia, Canada.
5
Immunology Laboratory, Vancouver General Hospital, Vancouver, British Columbia, Canada.
10
Institute for HEART + LUNG Health, Vancouver, British Columbia, Canada.
12
Queensland Facility for Advanced Bioinformatics and Institute for Molecular Bioscience, 13Queensland Diamantina Institute,
Translational Research Institute, The University of Queensland, Brisbane, Australia.
2

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GENOMICS AND PROTEOMICS IN KIDNEY TRANSPLANT

The analysis of high throughput omics approaches generates large amounts of data that require significant statistical
and computational breakthroughs to decipher complex biological systems. Several statistical approaches have been
proposed in the literature for the integration of two or more
high-throughput data sets. These include projection-based
multivariate approaches for biological exploration and ensemble classifiers for biomarker development and medical
decision making (Gunther et al., 2012; Le Cao et al., 2007).
Ensemble classifiers combine separately developed,
platform-specific classifiers using different combination rules
(Polikar, 2006). A popular rule is majority vote where the
predicted class is simply the one that is called by the majority
of classifiers in an ensemble. Integration of information from
different biological entities in ensemble classifiers happens
after platform-specific analyses are performed. This is in
contrast to projection-based multivariate approaches that are
discussed in this article, which integrate data from different
platforms at the analysis level.
Projection-based multivariate approaches are computationally efficient to handle large data sets, where the number
of biological features is much larger than the number of
samples, by projecting the data into a smaller subspace while
capturing the largest sources of variation in the biological
studies. During this statistical integration process, these
approaches produce a snapshot of the data and highlight
the largest sources of variation. However, when there is a
large number of biological entities to summarize each functional level, a projection of the data in a smaller subspace
might not be sufficient to extract relevant information (i.e.,
which genes, which proteins are relevant and are acting in
concert?).
In recent years, several variants of these statistical integrative approaches have been proposed to perform variable
selection and highlight those contributing to the largest variation in the data (Chun and Kelesx, 2010; Le Cao et al., 2008,
2009; Parkhomenko, et al., 2009; Waaijenborg, et al., 2008).
The approaches are based on the Partial Least Squares regression methodology (PLS), which enables the integration
of two data sets in a statistical sense: each data set is projected
into a smaller subspace so that the covariance or the correlation between both data sets is maximized. The improvement that these authors propose is to perform variable
selection, so that biological entities from both data sets that
are correlated with each other are directly extracted from the
methods. However, very few approaches have been proposed
so far to both integrate more than two data sets and to select
variables. Witten and Tibshirani (2009) proposed to concatenate all data sets with an appropriate weight applied to each
of them. Recently, a promising approach based on regularized generalised Canonical Correlation Analysis (rGCCA)
was proposed by (Tenenhaus and Tenenhaus, 2011) as a
generalization to the PLS approaches for more than two data
sets by maximizing the sum of the correlation in a pairwise
fashion between two data sets at a time, followed by a variant
that enables variable selection (Tenenhaus, et al., 2014.
In this article, we illustrate the usefulness and biological
relevance of selected multivariate approaches from Le Cao
et al., (2008; 2011) and Tenenhaus et al., (2014) on a clinically relevant biological example, which is an acute renal
allograft rejection study from the Biomarkers in Transplantation study. Kidney transplantation is a means to restore

683

kidney function in patients with kidney failure. Acute kidney

rejection after transplantation is a complication due to the
recipients immune responses to the foreign organ. Acute
kidney rejection is observed in *10% of renal transplant
patients. It is an important clinical problem with consequences for long-term graft survival. While current clinical
practice in post-transplant care and detection of acute rejection relies on additional sources of information such as tissue
biopsy and metabolic markers such as creatinine, the potential for blood-based biomarkers has been demonstrated using
data from a single genomics or proteomics platform (Freue
et al., 2010; Gunther et al., 2009). If one were able to diagnose acute rejection accurately based on a simple blood
sample draw, one could reduce the need for renal biopsies,
which are invasive, risky, and costly procedures. The main
aim of the kidney transplant study is to diagnose organ
transplant rejection using plasma protein and gene expression
data in blood samples from organ transplant patients. However, there exists a knowledge gap on how to best combine
multi-platform data sets to improve biological understanding
and accuracy of diagnosis. We show how the application of
projection-based multivariate statistical approaches can help
to better understand the information contained in the data sets
and to identify gene and protein signatures that can be used to
predict rejection status.
Material and Methods
The renal allograft study
Presentation of the study. Acute kidney allograft rejection was studied in a cohort of 40 patients, 20 of whom experienced an acute rejection (AR-patients) within 30 days
post-transplant and 20 controls who also received a transplant but did not experience an acute rejection for at least
6-months post-transplant (NR-patients). Blood samples were
collected at multiple scheduled time points and at the time
of suspected rejection for the AR-patients. Control samples from the NR-patients were time-matched with biopsyconfirmed AR-samples (i.e., for each AR-sample there was a
NR-sample from the same time-point). See Supplementary
Figure S1 for an overview of the blood sample collection).
Demographic and clinical information was considered in
Control-sample selection to minimize the impact of potential
confounders across the two groups (Table 1).

Table 1. Summary of Demographics for 20 AR

and 20 NR Patients Used in Study

Age at Transplant (s.d.)

Gender
Female
Male
Race
White
Asian
Other
Donor Type
Living donor
Deceased donor

AR (n = 20)

NR (n = 20)

45.74 (12.10)

49.04 (9.30)

6 (30%)
14 (70%)

8 (40%)
12 (60%)

19 (95%)
1 (5%)
0 (0%)

16 (80%)
3 (15%)
1 (5%)

13 (65%)
7 (35%)

13 (65%)
7 (35%)

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Description of the genomics and proteomics experiments. Peripheral blood samples were drawn into PAXgene

tubes (BD, Oakville, Canada) for genomics, and EDTA tubes

for proteomics analysis, stored at - 80C, and further analyzed on genomics and proteomics platforms. All 40 patients
had genomics and proteomics experiments run on blood
samples obtained at the same time. This prospective study
was conducted at the University of British Columbia. All
subjects provided written consent. The consent form and the
study was approved by the UBC-Providence Health Care
Research Ethics Board, and the UBC-Clinical Research
Ethics Board.
Genomics data. Samples were prepared and processed as
previously described (Gunther et al., 2009). Each sample was
then run on Affymetrix Human Genome U133 Plus 2.0 arrays
and scanned. Quality of arrays was assessed by visual inspection of boxplots, RLE, Nuse and M/A-plots that were
produced with the AffyPLM-package (Bolstad et al., 2005).
The 40 samples were combined with 253 additional Microarray samples from the larger Biomarkers in Transplantation
study, and all 293 samples together were background adjusted, normalized, and probe information was summarized
into probe-sets using the Robust Multi-Array Average
(RMA)-procedure from the RefPlus package in Bioconductor
(Harbron, et al., 2007). A prefilter step based on inter-quartile
range was applied to remove variables with little variation
across samples independent of sample class. Half of the
54,613 probe-sets with an IQR below the median IQR were
removed. The data are available through GSE46474.
Proteomics data. Plasma samples were collected in
EDTA tubes. Plasma was depleted of the 14 most abundant
proteins, followed by trypsin digestion and iTRAQ labeling.
Samples were then analyzed with iTRAQ MALDI-TOF/TOF
Mass Spectrometry and pre-processed as previously described
(Freue et al., 2010). ProteinPilot was used to assemble
identified peptide data into a list of identified proteins for each
iTRAQ run. These groups can contain more than one protein
(e.g., homologous or redundant proteins), or proteins belonging
to the same family that could not be distinguished based on the
available peptide. The Protein Group Code Algorithm (PGCA)
developed at the PROOF Centre linked protein groups across
different iTRAQ experiments. A protein group code (PGC)
dictionary was created with data from 444 kidney plasma
samples, including the 40 proteomics samples in this study.
The proteomics data set represents ratios of protein
abundance relative to pooled controls for all protein group
codes that were detected in at least one of the 40 samples.
Different from raw genomics data, proteomics data can have
missing values and a pre-filter was applied that required at
least 75% non-missing protein ratios across all 40 analysis
samples for the corresponding protein group (PG) to be included in the analysis. Missing values were imputed with
NIPALS and the resulting imputed data were log2 transformed (Wold and Lyttkens, 1969). The data are available in
Supplementary Material T1.
Statistical analysis
Overview. The exploratory statistical methods that were
applied in this report are projection-based methods, where the

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aim is to summarize the main characteristics of each data set.

From those approaches we can benefit from insightful builtin graphical outputs. These dimension reduction methods,
project the data into a much smaller subspace than the original space (which for omics data is typically highly dimensional), while capturing the largest sources of variation in
the data. The exploration and visualization of the structure of
the data in these new subspaces enable us to (1) assess if the
samples separate by phenotype (rejection status of the patients), (2) identify possible outliers, (3) reveal artificial
separation of data due to confounding variables, laboratory or
platform effects, and (4) identify relevant features in these
high dimensional data sets. To answer a wide variety of biological questions, we have performed two types of analyses
using several multivariate approaches. Figure 1 summarizes
the different analyses that were performed. The statistical
methods are described in the Supplementary Material M1.
Analysis of each data set separately. Principal Component Analysis ( Jolliffe, 2002) and Independent Principal
Component Analysis (Yao, et al., 2012) are both unsupervised techniques that do not take into account any prior
biological knowledge regarding the sample groups. This
preliminary step is particularly useful to understand the origin of the largest sources of the variation in the data, and the
similarities and differences that can be observed between the
independent samples given the expression or abundance data
sets. A supervised analysis was also performed with sparse
Partial Least Squares Discriminant Analysis (Le Cao et al.,
2011) by including prior knowledge regarding the groups of
patients (rejection status) in the analysis. The aim is to extract
and combine discriminative features (genes or proteins) that
best separate the different outcomes. Supervised analysis is
often performed for the identification of potential biomarkers
and the development of clinical classification models (Gunther et al., 2012).
Integrative analysis of two data sets. The second part of
the results presents a higher-level and novel analysis to integrate several sources of data simultaneously (genomics,
proteomics, but also rejection status of the patient). The aim
of these analyses is to gain additional insights that could not
be obtained by analyzing each data set alone, by identifying
biological features (genes, proteins) whose expression or
abundance is highly correlated across patients, in a supervised or unsupervised framework. Different integrative
modeling scenarios were investigated, building on the results
from the first part of the analyses. Sparse Partial Least
Squares regression (Le Cao et al., 2008) integrates genomics
and proteomics data in an unsupervised way, whereas sparse
Generalized Canonical Correlation Analysis (sGCCA, (Tenenhaus et al., 2014)) enables the integration of different
sources of data in a supervised framework. Different designs were considered to model the relationship between
the different data sets (subsequently called sGCCA-D1 and
sGCCA-D2). These integrative approaches are also part of
the projection-based methodologies and the resulting graphical outputs enable to visualize sample clustering, outlier
samples, and bias in the data.
Insightful outputs from the different approaches. The
statistical methods considered in this study are all based on

GENOMICS AND PROTEOMICS IN KIDNEY TRANSPLANT

685

FIG. 1. Summary of the

exploratory (Part 1) and integrative (Part 2) statistical
approaches applied to the
kidney transplant study. The
different sources of data are
represented by blocks, and
black lines between blocks
illustrate the relationship that
has been modeled between
the data sets.

similar principles to PCA and output components (also called

scores) which are linear combinations of the original variables. The weights in the linear combinations are determined
according to a given criterion, for example, maximizing the
variance in the data in PCA, or the covariance in PLS and
sGCCA methods. The components represent the data projected into a smaller subspace and lead to insighful graphical
outputs to highlight similarities between samples.
Other graphical outputs can also be obtained based on the
variable weights in the linear combinations to represent the
relationships between features when integrating two data
sets. In this output, a pair-wise similarity matrix is directly
computed from sPLS or sGCCA components on the basis of
the selected features only, instead of computationally intensive calculation of Pearson correlation coefficients between
each pair of variables in a high dimensional setting. We have
demonstrated in Gonzalez, et al. (2012) that the pair-wise
association values can be seen as a robust approximation of
Pearson correlation. Classical visualization outputs are then
obtained based on the pair-wise similarity matrix using relevance networks and Clustered Image Maps.
In addition, the sparse variants that we have applied enable
the selection of relevant features with respect to the criterion
that is maximized in each approach. In this article, we also
provide biological insight about the genes and proteins that
were selected with these multivariate approaches.
Results
Exploration and analysis of each data set
Unsupervised analysis of each separate data set

Principal Component Analysis. Three principal components explained 57.2% and 31.6% of the total variance on the
genomics and proteomics data respectively (Supplementary
Fig. S2). These rather low percentages give a first hint of the
amount of information that can be summarized from each

data source in a small number of components. The proteomics data seem to contain less relevant information than
the genomics data with three principal components. No clear
separation between the AR and the NR samples was observed
in the PCA sample plots (Supplementary Fig. S3). Interestingly however, most of the late samples ( > 2 weeks posttransplantation) clustered separately from the corresponding
early samples.
Independent Principal Component Analysis. IPCA was
able to separate the two groups of samples in a better way than
PCA and with less components (Fig. 2). The main separation
between AR vs. NR can be observed on the first component in
the genomics data but less so in the proteomics data. Most
importantly, the IPCA results support the earlier observation in
that the later samples cluster separately from the earlier ones
within their respective group (Supplementary Fig. S3).
Supervised analysis of each separate data set

Training and testing phases. As a way of assessing the

discriminative power of a selection of genes and proteins to
predict the rejection status of external patients, we divided
the original data set into a training and a testing set (see
Supplementary Material M1). The parameter tuning indicated an optimal selection of 90 probe-sets and 21 protein
groups (see Supplementary Fig. S4) Based on these selected
variables, the trained model could predict the class of new
test samples. Figure 3 shows that the separation between the
two groups is more accentuated for the genomics data than
for the proteomics data.
Final gene and protein selection. Final genomics and
proteomics models were trained on the full 26-sample
training set for the chosen parameters. The list of the selected
genes and protein groups can be found in Supplementary
Tables T2 and T3. Performance was assessed on the 14 test

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FIG. 2. IPCA sample representation. Samples were projected on the first two independent principal components for
the genomics data (a) and for the proteomics data (b).

samples by means of classification error, sensitivity, specificity, and AUC and compared with PLS-DA which includes
all variables in the model (Table 2). For both platforms, the
sparse version of PLS performed better than the non sparse
version. In the proteomics data, the PLS-DA method produced a high CE of 43% and an AUC of close to 0.5, which is
no better than a random classifier compared to a CE of 29%
(AUC = 0.76) with 21 selected proteins. The genomics classifiers performed better than the proteomics classifiers
(AUC = 0.90 with the selected 90 genes).
Integration of the two data sets

Even though genomics and proteomics data were extracted from blood samples, the two data sources are de-

rived from different compartments in the peripheral blood

(leukocyte cellular RNA in whole blood and proteins in
plasma). Previous analyses on the kidney biomarker in
transplantation data found that genomics and proteomics
biological rejection signals differ from each other, while
being consistent with the current understanding and pathogenesis of acute rejection injury (Freue et al., 2010;
Gunther et al., 2009). In an effort to unravel useful information about acute kidney rejection that could not be
obtained by analyzing each data set alone, we investigated
two integrative approaches, sPLS (Le Cao et al., 2008)
which is an unsupervised approach to extract common
signal between two omics data sets (i.e., correlated genes
and protein expression across patients) and its supervised
variant sGCCA (Tenenhaus et al., 2014).

GENOMICS AND PROTEOMICS IN KIDNEY TRANSPLANT

687

FIG. 3. sPLS-DA sample representations of the genomics

(a) and proteomics (b) data. sPLS-DA was first trained on
each data set [selection of 90 probe-sets in (a) and 21 proteins in (b)] on the 26 training samples (circles). For illustrative purposes, a second dimension was added but this
second component is not relevant to discriminate the two
classes. The testing samples (triangles) were then overlaid
on the plot based on the prediction given by the sPLS-DA
model. Late samples are shown with open symbols.

Unsupervised analysis of the integrated data sets with

sPLS

Variable selection. We ran a stability analysis as described

in Supplementary Material M1. The stability plot from Supplementary Figure S5 (b) shows that the selection of proteins was
more stable than the genomics data (probably due to the higher
number of variables in the genomics data set), with a small
number of protein groups consistently being selected. Our final
sPLS selection included 33 stable genes and 38 stable proteins
with a cutoff of 0.7 (see Supplementary Tables T4 and T5).
Graphical representations. Samples were projected on
the first two sPLS components for visualization purposes
[Supplementary Fig. S6 (a)]. Correlation circle plots are

graphical representations of the correlations between the selected variables and the sPLS components. These plots (whose
interpretation is detailed in Gonzalez et al., (2012) help to
visualize the correlation between the two types of selected
variables [clusters of features close to the circle of radius 1,
Supplementary Fig. S6 (b)]. Our observations were twofold:
first, we did not observe a very strong correlation between
genes and proteins; second, the sample representation does not
highlight a clear separation between the two groups of patients,
rather, the first sPLS dimension seemed to separate the samples according to the time of rejection (early vs. late).
Supervised analysis of the integrated data sets with
sGCCA. Two types of design were investigated with sGCCA.

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Table 2. Summary of Classification Performance for sPLS-DA and PLS-DA Classifiers Trained
on Full 26-Sample Training Set as Determined on a 14-Sample Testing Set (7AR and 7NR)

Genomics
Proteomics

Classifier

Number
of selected
variables

Classification
Error rate (CE)

Sensitivity

Specificity

Area under
curve

sPLS-DA
PLS-DA
sPLS-DA
PLS-DA

90
27,306 (all)
21
133 (all)

0.14
0.21
0.29
0.43

0.71
0.57
0.57
0.43

1
1
0.86
0.71

0.90
0.82
0.76
0.55

FIG. 4. sGCCA analysis with design 2. Sample representation on the genomics space (a) and the proteomics
space (b) for the first two dimensions.

GENOMICS AND PROTEOMICS IN KIDNEY TRANSPLANT

689

FIG. 5. Relevance networks of genes and protein groups

selected with sGCCA-design 1 (a) and with sGCCAdesign 2 (b). Only associations with an absolute correlation
greater than 0.5 are represented.

The number of components and features to choose for each data

set were tuned using the stability criterion described in Supplementary Material M1.
sGCCA-Design 1: All blocks are connected. The first
design is represented in Figure 1. The sample representations
of each data set highlighted a separation between the different
groups of patients, with a somewhat stronger separation in the
genomics space. Late samples are shifted along component 1
[Supplementary Fig. S9 (a) and (b)]. The stability analysis of
the variables selected produced higher frequencies compared
to sPLS (see Supplementary Fig. S7), which is understand-

able given the additional constraints posed in the sGCCA

model (the connection between blocks). Based on the stability analysis results, sGCCA selected 46 genes and 64
proteins that were stable. The genes and protein from this
analysis are further discussed in the biological interpretation
section.
sGCCA-Design 2: Genomics and the proteomics blocks
are connected separately to outcome status. Compared to
design 1, a much clearer separation between the two groups
of samples is obtained on the genomics side and is also better
on the proteomics side [Fig. 4 (a) and (b)], for a selection of

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Table 3. Gene Selected with sGCCA-Design 1

Probe Set ID

Gene title

234312_s_at
228758_at
202592_at
204495_s_at
1554016_a_at
235568_at
212463_at
208052_x_at

acyl-CoA synthetase short-chain family member 2

B-cell CLL/lymphoma 6
biogenesis of lysosomal organelles complex-1, subunit 1
chromosome 15 open reading frame 39
chromosome 16 open reading frame 57
chromosome 19 open reading frame 59
CD59 molecule, complement regulatory protein
carcinoembryonic antigen-related cell adhesion
molecule 3
carcinoembryonic antigen-related cell adhesion
molecule 3
cytohesin 4
DEAH (Asp-Glu-Ala-His) box polypeptide 34
dual specificity phosphatase 3
EF-hand domain family, member D2
EH domain binding protein 1-like 1
Fc fragment of IgG, high affinity Ia, receptor (CD64) ///
Fc fragment of IgG, high affinity Ic, receptor (CD64)
Fc fragment of IgG, high affinity Ib, receptor (CD64)
feline sarcoma oncogene
flotillin 1
flotillin 1
G protein-coupled receptor 97
GRAM domain containing 1A
hexokinase 3 (white cell)
interleukin 1, beta
integrin, alpha X (complement component 3 receptor 4
subunit)
leukocyte specific transcript 1
leukocyte specific transcript 1
leukocyte specific transcript 1
leukocyte specific transcript 1
microtubule associated monoxygenase, calponin and
LIM domain containing 1
metal-regulatory transcription factor 1
nuclear prelamin A recognition factor
netrin G2
outer dense fiber of sperm tails 3B
osteoclast associated, immunoglobulin-like receptor
phosphatidylglycerophosphate synthase 1
phosphopantothenoylcysteine decarboxylase
quiescin Q6 sulfhydryl oxidase 1
RAB24, member RAS oncogene family
RAB31, member RAS oncogene family
RAS guanyl releasing protein 4
S100 calcium binding protein A6
S100 calcium binding protein A9
SCO cytochrome oxidase deficient homolog 2 (yeast)
SH3-domain binding protein 2
SH3-domain binding protein 2
sialic acid binding Ig-like lectin 9
solute carrier family 12 (potassium/chloride
transporters), member 9
SWI/SNF related, matrix associated, actin dependent
regulator of chromatin, subfamily d, member 3
thymidine phosphorylase
uridine phosphorylase 1
zinc finger protein 438
SLAM family member 6
perforin 1 (pore forming protein)
ADP-ribosylation factor-like 4C
protein tyrosine phosphatase, non-receptor type 4
(megakaryocyte)

210789_x_at
219183_s_at
214017_s_at
201536_at
222483_at
221755_at
216950_s_at
214511_x_at
205418_at
208749_x_at
210142_x_at
220404_at
224807_at
205936_s_at
39402_at
210184_at
210629_x_at
211581_x_at
211582_x_at
215633_x_at
218376_s_at
205323_s_at
219862_s_at
233072_at
238327_at
1554503_a_at
219394_at
219066_at
201482_at
225251_at
217762_s_at
240862_at
217728_at
203535_at
205241_at
209370_s_at
211250_s_at
210569_s_at
220371_s_at
204099_at
204858_s_at
203234_at
229743_at
1552497_a_at
1553681_a_at
202206_at
205171_at

Gene symbol

Regulation in AR

ACSS2
BCL6
BLOC1S1
C15orf39
C16orf57
C19orf59
CD59
CEACAM3

Up
Up
Up
Up
Up
Up
Up
Up

CEACAM3

Up

CYTH4
DHX34
DUSP3
EFHD2
EHBP1L1
FCGR1A /// FCGR1C

Up
Up
Up
Up
Up
Up

FCGR1B
FES
FLOT1
FLOT1
GPR97
GRAMD1A
HK3
IL1B
ITGAX

Up
Up
Up
Up
Up
Up
Up
Up
Up

LST1
LST1
LST1
LST1
MICAL1

Up
Up
Up
Up
Up

MTF1
NARF
NTNG2
ODF3B
OSCAR
PGS1
PPCDC
QSOX1
RAB24
RAB31
RASGRP4
S100A6
S100A9
SCO2
SH3BP2
SH3BP2
SIGLEC9
SLC12A9

Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up
Up

SMARCD3

Up

TYMP
UPP1
ZNF438
SLAMF6
PRF1
ARL4C
PTPN4

Up
Up
Up
Down
Down
Down
Down
(continued)

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691

Table 3. (Continued)
Probe Set ID
205291_at
205758_at
210606_x_at
210972_x_at
211597_s_at
212656_at
216920_s_at
219034_at
221011_s_at
222482_at

Gene title
interleukin 2 receptor, beta
CD8a molecule
killer cell lectin-like receptor subfamily D, member 1
T cell receptor alpha locus /// T cell receptor alpha
constant /// T cell receptor alpha joining 17 ///
T cell receptor alpha variable 20
HOP homeobox
Ts translation elongation factor, mitochondrial
TCR gamma alternate reading frame protein ///
T cell receptor gamma constant 2
poly (ADP-ribose) polymerase family, member 16
limb bud and heart development homolog (mouse)
single stranded DNA binding protein 3

Gene symbol

Regulation in AR

IL2RB
CD8A
KLRD1
TRA@ /// TRAC ///
TRAJ17 /// TRAV20

Down
Down
Down
Down

HOPX
TSFM
TARP /// TRGC2

Down
Down
Down

PARP16
LBH
SSBP3

Down
Down
Down

Only the most differentially expressed between AR and NR for a p value < 0.05 (FDR correction).

41 genes and 60 proteins that were stable (Supplementary

Fig. S8). The genes and proteins from this analysis can be
found in Supplementary Tables T6 and T7.
Variable representations. The relevance network for design 1 shows many more correlations between genes and protein groups than the corresponding network for design 2
(Fig. 5). This is not surprising since design 1 implicitly required
correlations between the two data sets while design 2 did not
include this constraint. The same conclusions can be drawn
from the Clustered Image Maps (Supplementary Fig. S10).
Comparison between the different lists

Tables 5 and 6 show the overlap between genes and between proteins for all three integrative analyses. Also included for comparison are the 90 probe-sets and 21 protein
group lists returned by sPLS-DA (see Supplementary Tables
T2 and T3) when applied to the genomics and proteomics
data set separately. For the genomics data, we observed little
overlap between the lists for sPLS and the sGCCA models,

Table 4. Top Down- and Upregulated Proteins

in sGCCA-Design 1
geneSymbol

refseq

Regulation in AR

SERPING1
F13A1
APOA4
SERPINC1
F2
GC
SERPINA5
AFM
F13B
ADIPOQ
SERPINA4
PROC
SHBG
CST3
PON1
CANX
LBP
ARNTL2

NM_000062
NM_000129
NM_000482
NM_000488
NM_000506
NM_000583
NM_000624
NM_001133
NM_001994
NM_004797
NM_006215
NM_000312
NM_001040
NM_000099
NM_000446
NM_001024649
NM_004139
NM_020183

down

and some overlap with sPLS-DA, which is to be expected as

these methodologies focus on different characteristics of the
features. The concordance tables showed that there was more
overlap between the protein lists than the gene lists, which is
not surprising since the protein data set is much smaller than
the gene data set, and proteins tend to be consistently found
by the different approaches. Regarding sPLS-DA, the same
pattern as for the genes can be observed (i.e., sPLS-DA had
most overlap with sGCCA-D2 and least with sPLS).
Biological interpretation

In this section we particularly focused on the genes and

proteins returned by sGCCA-D1. To ensure a robust gene
analysis, we only considered the differential probe sets with a
median log2 expression higher than 6 (Welshs t-test, AR vs
NR, FDR < 0.05). This resulted in 51 genes upregulated in
AR and 17 genes down-regulated in AR (Table 3).
Using InnateDB (Lynn et al., 2008), we examined significantly up- and downregulated genes in order to identify overrepresented biological pathways or processes (Supplementary
Table T8). We identified that significantly upregulated genes in
AR compared to NR samples are representative of biological
processes involved in metabolic pathways and the hematopoietic cell lineage. Conversely, significantly downregulated genes
represent components of the T cell receptor (TCR) pathway and
downstream signaling pathways in nave CD8 + T cells.
Furthermore, performing an over-represented GO term
analysis of the upregulated genes using GOstat (Beibarth
and Speed, 2004) revealed that negative regulation of lymphocyte activation, negative regulation of T-helper 2 cell

Table 5. Overlap of Probe-sets in Gene Lists

Selected by sPLS, sGCCA-D1,
sGCCA-D2, and sPLS-DA
Genomics
up

sPLS
sGCCA D1
sGCCA D2
sPLS-DA

sPLS

sGCCA D1

sGCCA D2

sPLS-DA

33
5
0
0

5
46
3
5

0
3
41
24

0
5
24
90

The diagonal indicates the total number of variables selected by

each approach.

 NTHER ET AL.
GU

692

Table 6. Overlap of Protein Groups

in Protein Lists Selected by sPLS, sGCCA-D1,
sGCCA-D2 and sPLS-DA
Proteomics

sPLS

sGCCA D1

sGCCA D2

sPLS-DA

sPLS
sGCCA D1
sGCCA D2
sPLS-DA

38
35
23
8

35
64
43
11

23
43
60
17

8
11
17
21

The diagonal indicates the total number of variables selected by

each approach.

depletion of T lymphocyte-related processes are present in

ARs patient samples, as compared to NRs samples.
We also examined the proteins in this network that correlated with the genes addressed above (Table 4). Most notably, among the 13 most significantly downregulated proteins
there are three members of the serpin (serine protease inhibitor) family of proteins, namely SERPINA5, SERPINC1,
and SERPING1. These blood plasma proteins are involved in
regulation of blood coagulation and complement cascades
and inflammation.
Discussion

differentiation, and negative regulation of B cell apoptotic

process (Supplementary Table T9) are the most significant
biological processes represented by these genes. These biological processes correlate with the downregulated lymphocytemediated pathways revealed by the over-represented pathway
analysis described above. In addition, upregulated biological
processes include the positive regulation of interleukin-6
production, leukocyte chemotaxis, actin cytoskeleton reorganization, regulation of integrin biosynthetic processes,
regulation of cytokine production, and response to wounding,
fever generation, and phagocytosis. Together these biological
pathways are representative of immune responses, including
systemic inflammation and innate immune activities.
To examine the modulated genes across various cell
and tissue types, we took advantage of the gene enrichment
profiles created by Benita et al., (2010). Interestingly, both
the significantly up- and downregulated genes showed blood
cell specificity, as shown in Figures 6 and 7. Upregulated
genes are significantly enriched in myeloid cells, especially
neutrophils, compared to other blood cell types and various
other tissues. Conversely downregulated genes appear to be
significantly enriched in T-lymphocytes. Overall, these results reveal that increases in innate immune responses, neutrophil activities, and accumulation in the system, and

In an effort to explore relationships between genes and

proteins in acute kidney rejection, we have applied recently
developed variants of multivariate projection-based approaches to genomics and proteomics data sets from kidney
transplant patients that experienced an acute rejection event
versus nonrejecting controls.
The unsupervised analyses PCA and IPCA pinpointed an
interesting phenomenon regarding the separation between
the early and late samples, which has also been observed in a
related study that focused on longitudinal genomics data
only (Shin et al., 2014). In our study, the time of rejection was
defined as early (within 2 weeks post-tx) and late (more
than 2 weeks post-tx) based on input from clinicians. Most
acute rejection episodes in renal transplant patients in our
Vancouver, Canada cohort happened within the first 2 weeks
post-transplant. This is not necessarily true for other countries
where different medication regimens may be used pre- and
post-transplant. Australia and India show a similar behavior,
while numbers reported for the USA indicate that acute renal
rejection episodes occur later. It should be stressed that both
early and late rejection samples represent biopsy-confirmed
acute rejection and, as such, are phenotypically different from
the non-rejection samples. The observed clustering of late AR
with early NR in analysis sGCCA-D1 highlights the importance

FIG. 6. Gene enrichment profile of upregulated genes in sGCCA-D1. Red cells indicate genes that are enriched (known
to be highly expressed) in the corresponding tissue cell-types.

GENOMICS AND PROTEOMICS IN KIDNEY TRANSPLANT

693

FIG. 7. Gene enrichment profile of downregulated genes in sGCCA-D1. Red cells indicate genes that are enriched
(known to be highly expressed) in the corresponding tissue cell-types.

of the time of rejection relative to transplantation date, rather

than a rejection signal. It is possible that transplantation surgery and accompanying immunosuppressive therapy can
cause responses in the immune system that are likely to be
picked up in gene expression experiments. These changes
then overlay any acute rejection signal at times close to
transplantation.
A better separation between the two groups of samples was
observed in IPCA than in PCA. IPCA is a combined variant
of both ICA and PCA approaches. In previous studies, IPCA
was found to summarize the information in a smaller number
of components than PCA [see (Yao et al., 2012) for more
details]. The separation was not clear enough for definite
conclusions and led us to investigate supervised approaches
instead.
Genomics- and proteomics sPLS-DA classifiers were able
to separate AR and NR groups. The genomics classifier based
on 90 probe-sets performed better than the proteomics classifier based on 21 protein groups (Supplementary Tables T2
and T3). The predictive ability of the genes was higher than
the proteins, a fact that might not come as a surprise considering that iTRAQ technology is a semi-quantitative approach and subsequent estimate of protein abundance is noisy
and error-prone. Other technologies such as MRM will likely
work better but are typically targeted approaches that are
useful when a small list of candidate protein markers is available.
The overlap between the different lists of genes and proteins selected by the various multivariate approaches are
not surprising from a modeling perspective. As expected,
we observed large overlaps between supervised approaches
(sPLS-DA and sGCCA-D2) and approaches that focus on the
maximisation of the covariance between the genomics and
proteomics data sets (sPLS and sGCCA-D1). sPLS solely
focuses on maximizing the covariance between the omics
data sets, whereas sGCCA-D1 focuses on maximizing the
covariance between the omics as well as discriminating the
groups of samples, and sGCCAD2 focuses on the discriminative properties of the omics data to separate the groups of
samples. The approaches sPLS and sGCCDA-D1 did not
display a clear separation between AR and NR samples which
points towards the hypothesis of a lack of relationship between the genomics and the proteomics data.
Even though sGCCA-D2 was expected to find a better
separation between the AR and NR groups than sGCCA-D1

due to fewer constraints, this does not necessarily mean that

sGCCA-D2 has a better performance than sGCCA-D1 when
tested in an independent cohort. Design 2 allows selection of
features in a less constrained way but might include noisy
features that happen to have good individual discrimination
ability. We should note that for biomarker studies that go
beyond exploration, validation in an independent testing set
is required. Studies in which genes and proteins are integrated
for better understanding of underlying biology as presented in
this article would also benefit from validation studies in the
future.
We found that among the most downregulated proteins in
the plasma in AR were SERPINA5, SERPINC1, and SERPING1.
These proteins are known to have anti-inflammatory functions (Eror et al., 1999) through the inhibition of blood
coagulation factors as well as complement components. It
follows that the downregulation of these serpin family proteins in AR patient samples might be an indication of increased systemic inflammation and vascular permeability.
This proposal correlates with the known activities of the
genes identified in this network discussed above.
It has been shown that ischemic reperfusion tissue injury
might prevent immunosuppression-medicated acceptance of
allograft tissue and neutrophil accumulation and activity is
linked with the inhibition of the graft acceptance (de Perrot,
et al., 2003; Kreisel, et al., 2011a, b). The overall upregulation of inflammation and innate immune responses, including
neutrophil activation and accumulation, that we observe in
AR patient samples as compared to NR sample might
therefore be due to ischemic reperfusion tissue injuries from
the kidney transplantation.
It has also been shown that the complement activation is
associated with ischemic tissue injuries that result in the
production of a number of inflammatory mediators. For instance, C1 inhibitor, SERPINC1, is found to have protective
activities on ischemic injuries (Buerke, et al., 1995) that
treatment of intestinal ischemic injuries with C1 inhibitor
limits tissue damages and improves survival by inhibition of
complement activation as well as inhibition of neutrophil
infiltration, which leads to a reduction in the local inflammatory response (Lu et al., 2008). Also, complement depletion is shown to prevent neutrophil recruitment in a model
of myocardial ischemia (Buerke et al., 1995). The downregulation of SERPIN proteins in AR patients and the

694

potential decrease in complement activation as a consequence might therefore indicate reduced protective effects of
these proteins on ischemic tissue injuries.
Alternatively, downregulation of lymphocyte activities
in AR patient samples might be due to uremic conditions
brought on by kidney failure, as is implied by the increase in
patient creatinine levels. The association between uremia and
depletion of lymphocyte activities and proliferation is well
documented (Hauser et al., 2008; Kato et al., 2008; Nakai,
et al., 1992).
Conclusions

The availability of multiple omics data sets that measure

different biological properties of the same sample is becoming more common. As a consequence, there is a need for
statistical analysis tools that are able to integrate and utilize
information from all data sets beyond combination of results
from analyses applied by each omics data set separately. We
have applied unsupervised and supervised methods to explore and integrate genomics and proteomics data to determine which genes and proteins play important roles in acute
renal allograft rejection. Components along which groups of
interest are separated were further investigated to understand
which genes or proteins contribute most to that separation.
The exploratory unsupervised methods highlighted an interesting phenomenon regarding the time of rejection relative
to transplantation date. The supervised sPLS-DA method was
able to select a panel of diagnostic biomarkers that showed
good performance in classifying our test samples. We have
applied recently developed integrative methods that achieved
a clear separation of the two sample groups while selecting
promising genes and proteins candidates for the detection and
monitoring of acute kidney transplant rejection.
Acknowledgments

This work was supported by a Burroughs Wellcome Fund

Collaborative Research Travel Grant for Oliver Gunther who
visited the Queensland Facility for Advanced Bioinformatics
(QFAB) in Brisbane, Australia for 6 weeks in 2011. The work
of KALC is supported, in part, by the Wound Management
Innovation CRC (established and supported under the Australian Governments Cooperative Research Centres Program) and the Australian Cancer Research Foundation
(ACRF) for the Comprehensive Cancer Genomics Facility at
The University of Queensland Diamantina Institute.
Author Disclosure Statement

The authors declare that there are no conflicting financial

interests.
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Address correspondence to:

Dr. Kim-Anh Le Cao
The University of Queensland Diamantina Institute
The University of Queensland
Translational Research Institute
Brisbane, QLD 4102
Australia
E-mail: [email protected]