Computers in Biology and Medicine 43 (2013) 20532062

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Computers in Biology and Medicine

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Support vector machine algorithms in the search of KIR gene

associations with disease
Juan C. Cuevas Tello a, Daniel Hernndez-Ramrez b, Christian A. Garca-Seplveda b,n
a
Facultad de Ingeniera, Universidad Autnoma de San Luis Potos. Av. Dr. Manuel Nava No. 8, Zona Universitaria, ZC 78290, San Luis Potos, Mxico
b
Laboratorio de Genmica Viral y Humana, Facultad de Medicina, Universidad Autnoma de San Luis Potos, Avenida Venustiano Carranza #2405, Colonia
Filtros las Lomas, CP 78210, San Luis Potos, Mxico

art ic l e i nf o a b s t r a c t

Article history: Killer-cell immunoglobulin-like receptors (KIR) are membrane proteins expressed by natural killer cells
Received 7 May 2013 and CD8 lymphocytes. The KIR system consists of 17 genes and 614 alleles, some of which bind human
Accepted 30 September 2013 leukocyte antigens (HLA). Both KIR and HLA modulate susceptibility to haematological malignancies,
viral infections and autoimmune diseases. Molecular epidemiology studies employ traditional statistical
Keywords: methods to identify links between KIR genes and disease. Here we describe our results at applying
KIR articial intelligence algorithms (support vector machines) to identify associations between KIR genes
Computational biology and disease. We demonstrate that these algorithms are capable of classifying samples into healthy and
NK cells diseased groups based solely on KIR genotype with potential use in clinical decision support systems.
Articial intelligence
& 2013 Elsevier Ltd. All rights reserved.
Immunogenetics

1. Introduction
Killer-cell immunoglobulin-like receptors (KIR) are membrane
bound proteins expressed by natural killer (NK) cells and a small
subset of CD8 lymphocytes. NK cells are peripherally circulating
lymphocytes and key participants of innate immune responses to
viral infections and tumours. In contrast to the rearranging
antigen-specic receptors expressed by lymphocytes of the adap-
tive immune system, the KIR gene repertoire is genetically
determined and remains unchanged throughout life [1,2]. To date
17 KIR genes have been shown to exist, all of which exhibit allelic
polymorphism. KIR genes are organised within human chromo-
some 19q13.4 as physically contiguous strings known as haplo-
types [3]. A KIR haplotype is composed of centromeric and
telomeric halves known as haplotype motifs. All known KIR
haplotypes contain the four framework genes KIR3DL3,
3DP1,

2DL4 and
3DL2 but exhibit variation in the content of other
KIR genes (KIR2DL1,
2DL2,
2DL3,
2DL5A,
2DL5B,
2DS1,

2DS2,
2DS3,
2DS4,
2DS5,
2DP1,
3DL1 and
3DS1) [4].
KIR gene content diversity has recently been shown to arise from a
limited number of haplotype motif combinations (four centro-
meric and two telomeric motifs) [5].
KIR genes encode for two (2D) or three (3D) extracellular
domain proteins which can transduce activating (S) or inhibitory
n
Corresponding author. Tel.: 52 444 1135 860.
E-mail addresses: ca.garcia.s@gmail.com,
christian.garcia@uaslp.mx (C.A. Garca-Seplveda).
(L) signals upon binding their cognate ligands. The balance and
integration of these signals modulate NK cell cytotoxicity and
cytokine release [6,7]. Some KIR proteins recognise HLA class I
molecules and by doing so modulate the activity of NK cells [8,9].
KIR proteins interact with classical HLA class I ligands such as HLA-
A (KIR3DL2), HLA-B (KIR3DL1 and
3DS1) and HLA-C (KIR2DL1,

2DL2,
2DL3 and
2DS2) as well as with non-classical HLA
ligands such as HLA-G (KIR2DL4) [811]. The ligands of the
remaining KIR proteins have yet to be determined.
Virus-infected and malignant cells downregulate their expression
of HLA in an attempt to evade recognition by the adaptive immune
system. NK cells sense this loss (loss of self) through KIR and respond
vigorously by directly killing the infected or malignant target cells or
by releasing cytokines which recruit other immune cells [12,13].
Some KIR genes have been associated with increased susceptibility or
resistance to infectious, autoimmune and metabolic diseases [1419].
NK cells were initially identied by their ability to spontaneously kill
tumour cells without prior sensitisation [2022]. Historical studies of
the immunogenetic factors that determine clinical outcome in
patients subjected to haemopoietic stem cell transplantation (HSCT)
for haematological malignancies were the rst to highlight the
clinical relevance of KIR genes in antitumour responses [23].
The rst study to suggest such an association described a potent
graft-vs.-leukaemia effect arising from predicted NK cell alloreactivity
in the graft-vs.-host direction amongst patients subjected to HSCT for
leukaemias [24]. Many other studies published since describe KIR
gene associations with anti-tumour effects and post-transplant
clinical endpoints [2531]. Differences in patient demographics,
clinical management, the preferred transplant modality and KIR

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http://dx.doi.org/10.1016/j.compbiomed.2013.09.027
2054 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062

typing method have largely contributed to the heterogeneity of the Table 1

ndings. In addition, NK cell anti-tumour activity has been well Clinical data for the haematological cohort.
demonstrated to occur in vitro against a wide variety of haematolo-
n %
gical malignancies [23,32]. In all, these ndings support the notion
that KIR enable NK cells to play a crucial role at conferring resistance Gender Male 23 53
to certain haematological tumours [27,33,34]. Female 20 46
A proper assessment of the functional relevance of KIR genes in Diagnosis Chronic myeloid leukaemia 25 58
Hodgkin's lymphoma 18 42
human diseases should also take into account the allelic diversity of B symptoms Present 30 70
both KIR and HLA and the different afnities of their interaction [35 Absent 13 30
37]. Therefore, the daunting task of identifying associations between ECOG 0 3 7
KIR genes and disease, and of devising clinical algorithms that consider 1 16 37
2 20 46
the high biological complexity of KIR and HLA genes, is more easily
3 3 7
dealt with through computers and articial intelligence algorithms. 4 1 2
Support Vector Machines (SVM) were introduced by Cortes and
Vapnik as support-vector networks and the term widely dissemi-
nated by Cristianini and Shawe-Taylor [38,39]. SVM arose from the
machine-learning community's efforts towards developing articial Table 2
intelligence strategies for complex problems. Authors involved in Data encoding matrix exemplifying the distribution of articial-data-1.
statistical learning and kernel machine development have previously
g1 g2 g3 g4 g5 g6 g7 g8 ... g23 d1
described SVM's with greater detail [40,41]. SVM applications have
mainly focused on classication and regression problems. In classi- p1 0 0 1 0 0 1 1 0 ... 1 1
cation the goal is to produce a generalisable mathematical function p2 0 1 0 0 1 0 0 1 ... 0
1
capable of distinguishing between different classes. This function is p3 0 0 0 0 1 1 1 1 ... 1 1
... ... ... ... ... ... ... ... ... ... ... ...
inferred from available examples (training data) and then put to use p343 1 0 0 0 0 0 0 0 ... 1
1
on untested examples (test data). Regression applications, on the
other hand, produce a mathematical model which best describes the
behaviour of certain datasets so as to infer future trends (i.e., accordance with state and national ethics regulations and lacking
population growth prediction). SVM's excel over other classication personal identifying information so as to ensure patient/donor
algorithms in large part due to their capacity to perform multivariate condentiality.
classications in a non-linear manner and in n-dimensional space
(see below). This advantage allows several layers of complexity to be 2.2. KIR genotyping and encoding
taken into account, such as those typical of KIR, HLA and other
complex genetic systems. SVM are currently regarded state-of-the- KIR gene content was determined using a locally developed
art classication algorithms [40,41]. sequence specic priming polymerase chain reaction (SSP-PCR)
This paper summarises our experience at developing and apply- genotyping technique capable of detecting the presence or absence
ing SVM articial intelligence algorithms to identify associations of of each of the 17 genes [43]. Our SSP-PCR approach does not
KIR genes with disease in a cohort of patients with haematological currently enable us to distinguish between KIR2DL5A and B, the
malignancies. Our innovative use of genetic algorithms for the position of genes within the haplotype or gene copy number
optimisation of the SVM parameters allows for greater freedom from variations. PCR amplicons were resolved in 1.5% agarose gels and
human interference to be achieved. We consider that this innovative digitally documented after ethidium bromide staining. KIR haplotype
approach will prove valuable at identifying KIR gene associations in motifs (cA01, cB01, cB02, cB03, tA01 and tB01) were deterministically
other molecular epidemiological studies and aid in the development inferred from genotyping results as described with greater detail in
of clinical decision support systems (DSS). the original publication [5]. KIR genotyping information was encoded
for each sample as strings of binary values indicating presence (1) or
absence (0) of the 16 genes, a KIR2DS3/S5 gene combination and the
2. Materials and methods six haplotype-motifs for each of the 343 samples. These strings were
,
consolidated on a tab delimited text le as g g1, g2, g3 g23. In this
2.1. Study population text le KIR data for each of the samples p1 to p343 was arranged in
columns g1 through g23. In addition each data entry included a health
Three hundred unrelated blood-donor samples comprising the status variable dened as d1 which was1 in samples obtained from
Mexican Genomic DNA Reference Collection (MGDC-REF) were individuals having a haematological malignancy and
1 in healthy
used as healthy controls for this study. This Mexican mestizo donors, as shown in Table 2.
reference population included 135 (45%) males and 165 (55%)
females aged between 19 and 38 yr (median of 24) of which 75% 2.3. Articial data
were residents of the city of San Luis Potos and 25% were
residents of rural areas of this Mexican state. DNA samples were Two different articial datasets were used to assess the perfor-
extracted from blood-bank discarded leucocyte concentrates mance and establish baseline classication error plots for the SVM.
referred to us by Hospital Central Dr. Ignacio Morones Prieto The rst dataset (Articial-Data-1) was composed of 343 randomly
according to previously published protocols [42]. A more detailed generated KIR genetic trait strings which include KIR genotypes
description of the KIR features present in this reference population which were neither biologically relevant nor subjected to any
is given in the original publication [43]. In addition, 43 DNA haplotypic constraint. This dataset was used to evaluate the cap-
samples obtained from patients with haematological malignancies ability of the SVM at identifying gene associations where none would
(25 with leukaemia and 18 with lymphomas) referred to us by the be expected. KIR genotypic data was randomly generated (simulated)
Haematology Department of Hospital Central Dr. Ignacio Morones to produce an articial population of 343 samples and 23 variables
Prieto were included. Clinical information for the haematological (so as to maintain a similar size to that of the real dataset). To further
cohort is shown in Table 1. All samples were provided to us in help with dataset comparisons, the number of healthy articial
 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062 2055

individuals in this dataset were limited to 300 and the total number
of diseased samples was kept to 43. One hundred of these Articial-
Data-1 sets were assessed through SVM to generate average classi-
cation error, sensitivity and specicity plots.
A second data set (Articial-Data-2) was used to evaluate the
performance of the SVM at identifying KIR gene associations on
biologically sound KIR genotypes in which the health status variable
(d1) was randomly assigned. For this second dataset, the KIR
genotypes present in MGDC-REF were used to produce an articial
population of 343, 300 of which were randomly assigned a healthy
d1 variable and 43 a diseased variable (to maintain real dataset
proportions). Again, 100 different articial datasets were used to
generate average classication error, sensitivity and specicity plots.
2.4. Support vector machine and auxiliary algorithms
All SVM simulations were carried out using Matlab Support Vector
Machines toolbox for Classication and Regression [44]. In Fig. 1A, a
linear function separates four data points into two clearly distinct
classes (dark and open circles). This gure represents a simple
problem in which the data is linearly separable, a task that is easily
resolved by a linear classication algorithm like the Perceptron [45].
The line representing this function is located a distance between the
lower left dark circle and the lower right open circle, a distance that is
referred to as the classication margin [46]. In linear classication
problems, margin optimisation is essential to allow for the
proper separation of data points. However, when the classical XOR
problem is faced with linearly non-separable data points, the classi-
cation task becomes impossible for simple linear classication
algorithms (Fig. 1B left panel). One way in which this XOR problem
can be tackled is through SVM, where each data point is mathema-
tically transformed into a feature space () so as to allow for linear
classication (Fig. 1B right panel). In SVM, classication margins are
extended to linearly non-separable data through a soft margin (C), a
trade-off between maximising the margin and misclassifying data
points. To generate this soft margin the SVM rely on a kernel trick,
which together with margin maximisation, allows for the classication
of non-linear data [39,41,46]. The kernel trick allows non-linear data
distributions to be linearly separable by transforming them to a high-
dimensional feature space. Fig. 2 graphically demonstrates the perfor-
mance of a SVM non-linear classication algorithm at solving such
XOR problem.
,
In our approach, the mapping of g into required the use of a
kernel mapping function. We employed a Gaussian kernel known as
a radial basis function (RBF) kernel. However, the use of the RBF
kernel requires two parameters to be optimised and set: the width of
Gaussian functions (s) and the trade-off variable (C) that controls the
trade-off between misclassication and the size of the SVM margin.
Traditionally SVM optimisation relies on evaluating classication
performance with different s and C value combinations through a
labour intensive, empirical, trial and error approach.
Fig. 2. Non-linear classication using SVM for linearly non-separable data points
representing the XOR problem.

Fig. 1. (A) Classication is possible using a linear algorithm for these four data points (where black circles can be seen as representing diseased patients and open circles a
healthy individual). (B) Kernel trick. Non-linearly distributed input data is easily linearly classied when transformed to a high-dimensional feature space.
2056 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062

These optimisation procedures are explained with greater 3. Results and discussion
detail in previous publications [46]. Instead of taking this
approach, we chose to search for the optimal s and C values by The diversity and frequency of KIR gene features amongst the
applying a genetic algorithm to the task. For this purpose we healthy study population is described with greater detail in a
employed the Genetic Algorithm Toolbox for MATLAB (The Math- previous publication [43]. The KIR genetic features of the haema-
Works, Inc. Massachusetts, USA)[47]. This approach allowed us to tological cohort were similar to those of the healthy population.
achieve near-ideal classication errors (as close to 0 as possible) However, statistically signicant differences in the carrier frequen-
with greater precision and efcacy. Genetic algorithms achieve cies of KIR2DL2, Group A haplotypes and A,B haplotypes were
this through articial evolution sub-algorithms, which are a observed (see below). KIR haplotypes and haplotype motifs could
stochastic global search and optimisation method based on the be resolved through our deterministic encoding approach for more
principles of biological evolution [48]. Briey, the genetic algo- than 98% of our healthy control's and 100% of our patients
!
rithm begins with an initial generation P 1 having 50 random (allowing for the inherent ambiguities mentioned in the original
chromosomes (pairs of s and C values) as dened by publication) [5].
! For SVM to classify data, they rst have to be taught to
P 1 s1 ; C 1 ; s2 ; C 2 ; s3 ; C 3 ; sm ; C m  recognise the genetic patterns associated with disease using a
small subset of the real data, which is known as training data.
This rst generation is then subjected to articial genetic
Training data is normally a small fraction of the main dataset,
operators such as selection, crossover, mutation and re-insertion
! ! ! whereas the remaining part of the main dataset that is scrutinised
(elitist strategy) to generate P 2 ; P 3 ; P n generations. The
with the SVM is known as the test data. The magnitude of the
tness (f) of each generation is tested by the SVM in the search of
! classication error produced by a SVM on test data after training
the P n producing the lowest classication error and highest
varies depending on the complexity of the underlying pattern that
sensitivity and specicity (minimum f). This process leads to the
is being searched for. Classication error is also inuenced by the
optimisation of SVM parameters through articial evolution. Those
size and proportion of the main dataset that is being used for
generations producing the smallest f were selected for further use
training. In general terms, the larger the training dataset is, the
on real data. The ID3 algorithm was originally introduced by
smaller the classication error will be on test data. Nevertheless,
Quinlan in 1983 and used for rule generation in expert systems
the use of large training datasets (Z95%) leads to undesirable
[49]. We employed the ID3 algorithm as a supplementary data-
function over-tting. Optimal SVM performance is achieved using
mining tool to generate decision trees from our real data set.
training dataset sizes ranging between 50% and 80% [40,45]. Initial
While SVM's are useful to model the relationship that exists
SVM simulations employed an input matrix composed of 35 KIR
between input and output data they are not capable of indicating
variables: 16 genes, 6 classical haplotype combinations (A/A, A/B,
which variables are more relevant during the classication pro-
B/B, all having A, all having B and Bx), 6 haplotype motifs and
cess. By using the ID3 algorithm we were able to visualise an
7 extended haplotypes. However, subsequent trials using different
approximation of the underlying genetic pattern being recognised
combinations of these variables in the matrix demonstrated
by the SVM in the form of a decision tree by using information
differences in SVM performance (data not shown). Final simula-
entropy. Improved versions of ID3 include C4 and C5 algorithms.
tions and those discussed herein were carried out using only the
A plot of the SVM classication error on test data was generated
limited matrix variables mentioned in the KIR genotyping and
at different training-data sizes in 5% increments for the three
encoding subsection of Materials and methods.
datasets. Plots of the sensitivity and specicity achieved by the
We evaluated the classication performance of our SVM algo-
SVM on test-data at different training-data sizes were also
rithm at classifying samples into healthy and diseased groups
produced. ID3 classier models for decision trees were generated
using three different datasets: (1) Articial-Data-1, (2) Articial-
with 5-fold cross-validation for three different training data sizes:
Data-2 and (3) Real Data. The Articial-Data-1 matrix was used to
30%, 50% and 75%. Overall specicity [true negatives/(true nega-
assess baseline performance of the SVM at identifying associations
tives false positives)] of the SVM algorithm was calculated for
between genes and disease where none should in theory exist.
each training dataset size increment along with overall sensitivity
Less than 5% of the 34,300 articial genotypes that were randomly
[true positives/(true positives false negatives)]. Receiver Operat-
generated using this dataset matched actual KIR haplotypes seen
ing Characteristic (ROC) curves were generated by plotting true
in human populations. The Articial-Data-2 matrix allowed us to
positive rates (TPR) against false positive rates (FPR), i.e. sensitivity
evaluate the performance of the SVM at dealing with biologically
vs. 1specicity, achieved at different training dataset sizes for
sound KIR haplotypes or genetic traits. However, these genetic
each of the three datasets [50]. When a discrete classier is
traits were randomly assigned a health status variable and as such
applied to a test set it yields a single confusion matrix (true
do not represent actual KIR haplotypes present in real patients.
positive rate vs. false positive rate) which is represented as a single
The optimal s and C values for each of these datasets were
ROC point [51]. Each ROC point evaluates the performance of the
calculated independently and using the entire datasets. A genetic
SVM on test data at different training data sizes.
algorithm enabled us to quickly determine the best s and C
parameter combination for this task. For Articial-Data-1 the best
2.5. Traditional statistical tests parameters were shown to be C 6.0144 and s 0.047435, while
for both the Articial-Data-2 and Real Dataset these were C
Carrier (phenotypic) frequencies for KIR genes, haplotypes, 0.087604 and s 0.47026. As expected, the classication error
motifs and genotypes were calculated by counting the number of plots shown in Fig. 3 (average classication error produced from
individuals bearing the trait and the percentage that they repre- 100 iterations for each dataset) followed a logical stratication of
sent in either the reference or study populations. KIR gene and results. Randomly generated KIR genotypes (Articial-Data-1)
haplotype carrier frequency comparisons between healthy con- produced high overall classication errors and real data produced
trols and diseased patients employed a two-sided Pearson's 2 or the lowest. The overall classication error plot for Articial-Data-2
Fisher's exact test along with multivariate logistic regression tests was located between the former two. SVM misclassication is
for independence using the SPSS Production Facility (version 16, dened as an incorrect prediction of the health status of a test
SPSS, Inc. Chicago, IL, USA), signicance being established at genotype based on the rules established from training data. These
p o0.05. could be test genotypes predicted to be healthy when they were
 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062
Fig. 3. Classication error plot on test-data for both articial and real data as a
function of different training-data set sizes.
actually diseased or vice versa. The mean classication error of our
SVM algorithm at optimal training dataset sizes (between 50% and
80% as mentioned previously) was of 12.1% (ranging from 11.7% to
12.5%) for Articial-Data-1, 9.96% (ranging from 9.1% to 10.7%) for
Articial-Data-2 and of 6.5% (ranging from 5.8% to 7.3%) for the Real
Data. Within the optimal training dataset size region, the SVM
algorithm manages to classify KIR genotypes correctly in 87.9% of
the Articial-Data-1, 90.1% of the Articial-Data-2 and in 93.5% of the
Real Dataset. For the three datasets, a statistical comparison of mean
classication errors revealed pr0.0001; using students-t test
throughout all comparisons. One immediate inference arising from
this distribution of classication error plots is that the SVM is capable
of identifying genetic patterns associated with health status. This can
only arise from a true association of KIR genes with susceptibility to
haematological malignancies, as the SVM manages to achieve lower
classication errors in the real data in comparison to the articial
datasets. If KIR genes were not associated with susceptibility or
protection to haematological diseases then the classication plot for
the real dataset would be expected to be higher and similar to that of
the Articial-Data-2 plot.
The SVM fails to achieve as low classication error on Articial-
Data-1 given the random nature of the KIR genotype strings used and
,
the absence of a true association between g and d1. The Articial-
Data-1 plot therefore represents the baseline performance of our
algorithm and its capacity of nding associations between genetic
patterns and health status where none are present. This is merely a
reection of the capability of SVM algorithms at identifying patterns
and, for simplicity, could be regarded as the maximum SVM
performance on random data and the curve to which all other data
sets should be compared. As one would expect, the SVM algorithm
performs even better when real KIR genotypes are used in spite of
the randomness with which the health status variable was assigned.
With the real dataset however, a true underlying biological pattern
exists and associates KIR genotype with outcome. This nding alone
provides further support to a critical role for KIR in early anti-tumour
responses. That NK cells play a crucial role in immune responses
against haematological malignancies has been well established [52
57]. However, little evidence exists of the mechanisms by which KIR
genes provide NK cells (or other lymphocytes) the capacity to
eradicate or tolerate malignant cells [58]. That classication error
reaches near-articial-data levels at training-data sizes below 20%
and beyond 80% is only a reection of a disproportionate distribution
of healthy to diseased cases at these dataset ratios (4:30 and 30:4,
respectively).
2057
Plotting sensitivity and specicity against training data size
allows the performance of the SVM classier to be further
assessed, see Fig. 4. Overall specicity of the SVM algorithm at
optimal performance range for Articial-Data-1, Articial-Data-2
and Real Data was of 100%, 97% and 96%, respectively. On the other
hand, overall sensitivity of the SVM algorithm at optimal perfor-
mance range for these same datasets was of 8.5%, 13.4% and 58.6%,
respectively. In all cases, SVM performance on real data was better
than that observed with Articial-Data-2. The authors consider
that the high overall specicity seen in the three datasets results
mainly from the abundance of healthy subjects. However, the fact
that sensitivity was much higher in the real data set in comparison
to the two articial datasets reects the algorithms ability to
correctly identify a genetic pattern associated with disease (see
Fig. 4 left panels). ROC curves generated from the specicity and
sensitivity plots for each dataset (at different training dataset
sizes) further illustrate this (Fig. 4, panels on the right). In these
plots the articial data sets produce ROC curves which are closer
to the reference diagonal dotted line that represents a random
guess strategy. Calculating area under the curve (AUC) for these
plots shows that higher values are present in the real dataset
(0.73570.073, mean 7SD) followed by the Articial-Data-2
(0.540 70.039) and Articial-Data-1 (0. 543 7 0.052).
Figs. 3 and 4 show the performance of the optimised SVM
classier and demonstrate the feasibility of applying this approach
to predict clinical outcomes based solely on KIR genotyping data
for diseases that have shown to be associated with these genes. It
is clear that the optimal performance of the SVM on Real Data is
achieved when using training data sizes that range between 30%
and 60%. Within this size range classication error is lowest (5.8%)
and sensitivity is highest (64.5%). A summary of the classication
errors, sensitivity, specicity, healthy subject number, true positive
and true negative rates achieved on real data is given in Table 3.
Comparison of the ROC curves and AUC calculations reveals that
those derived from articial dataset were closer to the random-
guess diagonal (AUC 0.5) than those derived from real data.
The SVM manages to classify Real Data samples by using n-
dimensional transformations of KIR genotyping data to generate a
linear plane capable of clearly distinguishing between healthy and
diseased status. As the visualisation of this n-dimensional feature
space escapes current graphical means of representation, the ID3
algorithm allowed us to indirectly generate binary decision trees
that highlight some of the most important KIR patterns being
recognised by the SVM algorithm. ID3 classier models for
decision trees were generated on real data using 5-fold cross-
validation for three different training data sizes: 30%, 50% and 75%
(see Fig. 5). As expected, our results show that ID3 decision trees
tend to become more complex as the training data size increases.
The decision tree generated using 30% of training data (panel A in
Fig. 5) requires only 6 decisions and 7 KIR genetic traits to classify
the remaining 70% of the dataset (test data) as healthy or diseased
with less than 5.8% classication error, a sensitivity of 61.3% and a
specicity of 99%. However, at a training dataset of 75% and at a
point where SVM performance is optimum, the decision tree
(panel C in Fig. 5) requires 8 decisions, 16 KIR variables and a
more complex tree topology to achieve the same, albeit with a
higher specicity (100%). Interestingly, the decision tree generated
at mid range (panel B in Fig. 5) exhibits features present in both
the rst and third tree. Traditional statistical comparison of KIR
carrier frequencies (contingency tables using Pearson's or Fishers'
exact test) revealed that KIR2DL2 was more frequent amongst
patients with haematological malignancy in comparison to the
healthy donors (77.8% vs. 40.3%, respectively; p r0.0001), Group A
homozygosity was less frequent (11.1% vs. 32%, respectively;
pr 0.0044) and A,B heterozygous haplotypes were more frequent
(86.7% vs. 58.7%, respectively; pr 0.0002). Although decision trees
2058 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062

Fig. 4. Sensitivity and specicity plots (left panels) and ROC curve plots (panels on the right) for the two articial datasets and the real dataset as a function of different
training-data set sizes.

generated at 50% and 75% training data did support the impor-
tance of KIR2DL2, classical KIR haplotype groups showed no
relevance. On the other hand, the importance of the centromeric
haplotype motif cB03 seen in these same trees did not prove to be
statistically signicant through traditional analysis. Also evident
from the three ID3 trees is that most of the decisions involved
either KIR genes or haplotype motifs rich in KIR genes having HLA-
C specicity: 85% of the decisions made at 30% training data, 80%
of those at 50% training data and 62% of those made at 75%
training data size. KIR genes having HLA-Bw4 specicity proved to
be relevant only after three of more HLA-C decisions had taken
place, which highlights the importance of this specicity in this
particular group of haematological diseases. Although our geno-
typing technique was not capable of distinguishing between
KIR2DL5A and -B, the ID3 tree generated at 75% training size
highlights the importance of this gene as suggested by other
authors [30,59]. A previous report had associated the KIR3DS1
2DL52DS52DS1 genotype with protection from Hodgkin's
 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062 2059

Table 3
Support vector machine performance summary on real data.

Training data (%) CE (%) Sens (%) Spec (%) Healthy (n) Diseased (n) True negative True positive

5 12.9 17.1 97.2 285 41 277 7

10 9.4 38.5 98.1 270 39 265 15
15 11 37.8 96.5 255 37 246 14
20 7.3 60 97.5 240 35 234 21
25 6.6 54.5 99.1 225 33 223 18
30 5.8 61.3 99 210 31 208 19
35 6.7 57.1 98.5 195 28 192 16
40 6.8 57.7 98.3 180 26 177 15
45 6.3 58.3 98.8 165 24 163 14
50 5.8 63.6 98.7 150 22 148 14
55 6.5 60 98.5 135 20 133 12
60 6.5 61.1 98.3 120 18 118 11
65 7.4 56.3 98.1 105 16 103 9
70 6.8 53.8 98.9 90 13 89 7
75 5.8 54.5 100 75 11 75 6
80 7.2 44.4 100 60 9 60 4
85 9.6 28.6 100 45 7 45 2
90 11.4 20 100 30 5 30 1
95 11.1 33.3 100 15 3 15 1

CE classication error, Sens sensitivity, Spec specicity.

Fig. 5. ID3 decision trees generated at three different training dataset sizes: (A) 30%, (B) 50% and (C) 75%.

Lymphoma [60]. Our description of a protective effect arising from nding. Given that KIR2DP1 is a pseudogene and that KIR2DL1 and
the presence of the cB03 motif (which harbours KIR2DL3,
2DL5,
2DL3 have such a high carrier frequency in our population, we

2DS5,
2DP1 and
2DL1 genes) is in agreement with this think that this protective effect might depend mainly from the
2060 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062
presence of KIR2DL5 and 2DS3/S5 locus, an effect that has also been
inferred by previous studies [59,60]The protective effect provided by
KIR3DS1 that has been reported by other authors was also partially
supported by our data [30,60]. However, in our results the 3DS1 gene
only provided a protective effect when observed in the absence of
2DL2 or 2DL5 genes (see Fig. 5, panel c). In a similar manner, we
found that KIR2DS1 gene had a protective effect when present in
genotypes not harbouring KIR3DL1 but also having KIR genes with
HLA-C2 specicity such as KIR2DL2,
2DS2 and
2DL3. These
results are in partial agreement with those published previously
[30,6062]. However, our results did not conrm previously pub-
lished ndings relating KIR2DS3 or the number of activating KIR
genes to haematological malignancy [59,63,64].
Based on these ndings we believe that KIR genes with HLA-C
specicity render either NK cells or KIR bearing T cells more capable
of detecting and eradicating malignant cells by providing them with
an activation-prone repertoire or by enhancing the detection of
higher levels of peptide-MHC class I complexes as has been
suggested for EBV associated Hodgkin's Lymphoma [60]. Finally, it
is the authors' belief that the protective effect identied by ID3 in the
form of the haplotype motif cB03 arises mainly from the KIR2DL3
and KIR2DS3S5 combination. This assumption is based on the fact
that the ligand for KIR2DL5 has yet to be resolved. This together with
the fact that some KIR2DL5 alleles are not expressed reduces the
likelihood of an important role for the KIR2DL3 and KIR2DL5
combination. In contrast, the extracellular immunoglobulin-like
domains of KIR2DL3 and KIR2DS3S5 exhibit high homology [65].
This notion is further supported by the fact (1) that neither KIR2DL1
nor
2DL3 are on their own important steps in the ID3 decision
processes, (2) that KIR2DL1,
2DL3,
2DL5 and
2DS3S5 are also
present in other haplotype motifs which were not identied as being
important such as cA01 for KIR2DL1 and
2DL3 combination, cB01
for the KIR2DL1,
2Dl5 and
2DS3S5 combination. This could be
interpreted as resulting in a redundant protective KIR repertoire that
renders KIR-bearing lymphocytes capable of both detecting the
absence of HLA-C1 allotypes resulting from tumour immune evasion
strategies or the presence of up-regulated HLA-C1 allotypes resulting
from increased MHC expression as has been suggested for certain
Hodgkin's lymphomas [66]. Although the molecular genotyping
method that we employed did not allow us to include information
on KIR gene number variations, high-resolution allele typing's, HLA
data and the position of genes within specic haplotype motifs, we
would not expect the SVM to encounter any difculties at tackling
the information once made available.
4. Conclusions
While the importance of KIR genes having HLA-C specicity has
also been suggested by the aforementioned leukaemia suscept-
ibility studies, our results provide a novel insight into the different
functional arrangements of genes that might be involved.
Although the size and heterogeneity of our study cohort together
with the lack of HLA typing data limits the clinical inferences that
can be made from our results, it sets an example for a different
way of analysing the clinical and functional relevance of complex
genetic systems. Furthermore, our results highlight the possibili-
ties of applying computational supervised learning methods to
analyse data and recognise patterns capable of being used in
clinical decision-making. The capabilities of articial neural net-
works at predicting acute graft-vs.-host disease in patients with
thalassaemia subjected to unrelated donor HSCT have been pre-
viously demonstrated [67]. As such we have developed an alter-
native and state of the art SVM approach to analyse KIR patterns
and provide a simple clinical DSS that incorporates such data. We
consider that this SVM approach will reveal previously unknown
associations with disease susceptibility and progression in future
applications. Current efforts are being focused at our laboratory on
applying this same approach in the search of KIR gene associations
in larger and more homogeneous disease groups such as HIV/AIDS,
CMV and A(H1N1) pandemic inuenza.
Conict of interest statement
None declared.
Summary
Killer-cell immunoglobulin-like receptors (KIR) are membrane
bound proteins expressed by natural killer (NK) cells and CD8
lymphocytes. To date 17 KIR genes and 614 alleles of these have
been shown to exist. The highly polymorphic genetic system o
Human Leucocyte Antigens (HLA) are ligands for some KIR proteins,
to date nearly 6000 alleles of HLA genes are known to exist. Tumour
cells such as those of leukaemia and lymphomas, downregulate their
expression of HLA in an attempt to evade recognition by the adaptive
immune system (i.e., CD8 lymphocytes). NK cells (part of the innate
immune system) sense this loss of HLA expression through KIR and
respond vigorously by killing the malignant target cells or by
releasing cytokines which recruit other immune cells to destroy
them. The daunting task of identifying epidemiologic associations
between diverse gene systems and neoplastic diseases is more easily
dealt with through computers and articial intelligence algorithms.
In this manuscript we describe our results at applying support vector
machine articial intelligence algorithms (SVMs) to identify KIR gene
associations in a cohort of patients with haematological malignan-
cies. A total of 300 clinically healthy unrelated blood donors (used as
controls) and 43 samples obtained from patients with haematologi-
cal malignancies (25 with leukaemia and 18 with lymphomas) were
genotyped for the 17 KIR genes using an in-house DNA-based PCR-
SSP approach. KIR genotyping strings and clinical outcome (healthy
or diseased) were encoded into a matrix le which was then
subjected to analysis through a SVM algorithm and the algorithm's
performance at classifying samples into healthy and diseased eval-
uated and compared to that achieved on an entirely random data
matrix (set 1) as well as to an additional articial data matrix (set 2)
generated using real KIR genotyping strings randomly assigned
healthy or ill status. As expected, the classication error plots
followed a logical stratication of results where the randomly
generated matrix (set 1) produced high overall classication errors
and real data produced the lowest, with the second articial dataset
lying in between these two. Our results provide further support
linking KIR genes with susceptibility to malignant diseases and an
alternative state of the art AI approach to the analysis of complex
genetic systems in epidemiology. We feel that similar approaches
will aid future molecular epidemiology studies as well as in devel-
oping clinical decision support systems.
Acknowledgements
The authors wish to thank Dr. Oscar Prez Ramrez and Dr.
Arturo Snchez Arriaga of the Haematology Service and Blood
Bank of Hospital Central Dr. Ignacio Morones Prieto for referring
the patient samples that made this work possible. To Dr. Daniel E.
Noyola of the Virology Laboratory, Facultad de Medicina Universi-
dad Autnoma de San Luis Potos for proofreading the manuscript.
This work was funded by Grants provided from Universidad
Autnoma de San Luis Potos (P/PIFI2009-24MSU0011E-12) and
Fondo Sectorial de Investigacin en Salud y Seguridad Social
 J.C. Cuevas Tello et al. / Computers in Biology and Medicine 43 (2013) 20532062
(SALUD-2009-01-115226). The study sponsor had no role in study
design, collection, analysis and interpretation of data.
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Juan Carlos Cuevas received a Masters degree in Computer Sciences (Articial
Intelligence) from Universidad Nacional Autnoma de Mxico and a PhD degree on
Computer Sciences and Articial Intelligence from Birmingham University, United
Kingdom. He is currently full-time research professor at the Faculty of Engineering
Universidad Autnoma de San Luis Potosi.
Daniel Hernndez-Ramrez is currently working towards his PhD degree at the
Human and Viral Genomics Laboratory of the Facultad de Medicina Universidad
Autnoma de San Luis Potosi, in San Luis Potosi, Mxico. His project relates to
dening the clinical relevance of HLA and KIR diversity on HIV infections amongst
Mexican mestizos.
Christian A. Garca-Seplveda received a Medical degree from Facultad de
Medicina, Universidad Autnoma de Coahuila, Mxico and a PhD degree from
University College London. He is currently a full-time research/professor and head
researcher at the Human and Viral Genomics Laboratory of the Facultad de
Medicina Universidad Autnoma de San Luis Potosi, in San Luis Potosi, Mxico.