Comparative multivariate analysis of codon and amino acid usage in three Leishmania genomes.

Abstract

Multivariate analysis of codon and amino acid usage was performed for three Leishmania species, including L. donovani, L. infantum and L. major. It was revealed that all three species are under mutational bias and translational selection. Lower GC12 and higher GC3S in all three parasites suggests that the ancestral highly expressed genes (HEGs), compared to lowly expressed genes (LEGs), might have been rich in AT-content. This also suggests that there must have been a faster rate of evolution under GC-bias in LEGs. It was observed from the estimation of synonymous/non-synonymous substitutions in HEGs that the HEG dataset of L. donovani is much closer to L. major evolutionarily. This is also supported by the higher dN value as compared to dS between L. donovani and L. major, suggesting the conservation of synonymous codon positions between these two species and the role of translational selection in shaping the composition of protein-coding genes.

Full text

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GENOMICS
PROTEOMICS &
BIOINFORMATICS
www.sciencedirect.com/science/journal/16720229
Article
Comparative Multivariate Analysis of Codon and Amino Acid Usage
in Three Leishmania Genomes
Nutan Chauhan, Ambarish Sharan Vidyarthi, and Raju Poddar*
Department of Biotechnology, Birla Institute of Technology, Mesra, Ranchi-835215, India
Genomics Proteomics Bioinformatics 2011 Dec; 9(6): 218-228 DOI: 10.1016/S1672-0229(11)60025-9
Received: May 02, 2011; Accepted: Oct 31, 2011

Abstract
Multivariate analysis of codon and amino acid usage was performed for three Leishmania species, including L.
donovani, L. infantum and L. major. It was revealed that all three species are under mutational bias and transla-
tional selection. Lower GC12 and higher GC3S in all three parasites suggests that the ancestral highly expressed
genes (HEGs), compared to lowly expressed genes (LEGs), might have been rich in AT-content. This also sug-
gests that there must have been a faster rate of evolution under GC-bias in LEGs. It was observed from the esti-
mation of synonymous/non-synonymous substitutions in HEGs that the HEG dataset of L. donovani is much
closer to L. major evolutionarily. This is also supported by the higher dN value as compared to dS between L.
donovani and L. major, suggesting the conservation of synonymous codon positions between these two species
and the role of translational selection in shaping the composition of protein-coding genes.
Key words: Leishmania, relative synonymous codon usage, multivariate analysis, hydropathy, aromaticity


Introduction 
Leishmaniasis, an infectious protozoal disease caused
by parasites belonging to the genus Leishmania, is
still one of the world’s most neglected diseases, af-
fecting mainly developing countries (1). L. major
causes the most common form of infection, cutaneous
leishmaniasis, while L. donovani and L. infantum are
associated with visceral leishmaniasis (2, 3), also
known as Kala-azar, in the Indian subcontinent, East
Africa, and Mediterranean regions (4). Despite the
continuous ongoing efforts in antileishmanial drug
discovery and development, there is no effective
medicine available so far. The results from current

*Corresponding author.
E-mail: rpoddar@bitmesra.ac.in
© 2011 Beijing Institute of Genomics. All rights reserved.
chemotherapeutic drugs available for the treatment of
Leishmania infection are not satisfactory (5). The
toxic nature of available drugs and the tendency of
Leishmania to become resistant reflect the need for
discovery of more effective antileishmanial agents (5).
Therefore, there is an urgent need to understand the
biology of these three Leishmania pathogens. The
published genomic details of L. infantum (6) and L.
major (7) show that the average GC content was
around 59% for both of them. The whole genome of L.
donovani has yet not been sequenced, but sequences
of some genes and proteins are available online.
Many genes demonstrate a non-random selection of
codons in their protein-coding regions. For any given
protein we can distinguish at least two sources of bias
in codon usage. The first, “amino acid preference”, is
the uneven amino acid composition of typical proteins,

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
Genomics Proteomics Bioinformatics 2011 Dec; 9(6): 218-228 219
i.e., some amino acids are used far more frequently
than others (8). The second is that once an amino acid
has been chosen, there are generally preferences for
the use of certain codons. Relative synonymous codon
usage (RSCU) and relative amino acid usage (RAAU)
are used to measure the non-random usage of specific
amino acid. Genes with strong codon bias appear to
be expressed at a higher level compared to other
genes. Biased codon usage may result from a combi-
nation of several factors, namely, biases in the pattern
of mutation (9), or translational selection (10) among
synonymous codons. Within-species heterogeneity in
codon usage has been most clearly elucidated in E.
coli (11). The major trend includes a strong bias to-
wards a particular subset of codons in highly ex-
pressed genes (HEGs) and more even codon usage in
lowly expressed genes (LEGs) (12-14). Our compara-
tive multivariate analysis of codon and amino acid
usage patterns in Leishmania species will provide an
insight into the divergence and compositional simi-
larities within and across their genomes and may lead
to a better understanding of the biology of the para-
sites and the development of more effective drug
treatments.
Results
Major sources of RSCU variation in the three
Leishmania species
Correspondence analysis (COA) was used to explore
the variation of RSCU values in the genes from L.
donovani, L. infantum and L. major. After plotting
genes in 59-dimentional hyperspace, according to the
usage of the 59 synonymous sense codons (stop
codons and codons with one-to-one mapping to amino
acids, i.e., Met, Trp were excluded), COA identifies a
series of new orthogonal axes accounting for the
greatest variation among genes. The coordinate of
each gene on each new axis and the fraction of the
total variation accounted for by each axis is generated
by COA. Axis 1 and Axis 2 indicate the major trends
of variations among genes. Axis 1 accounts for 31.5%,
15.7% and 17.2% of the total variations for RSCU in
L. donovani, L. infantum and L. major, respectively
(Table 1). In all cases, GC3S (GC content at synony-
mous codon sites excluding ATG for Met and TGG
for Trp) and NC (effective number of codons) exhib-
ited strong correlation with Axis 1. The correlation
between GC3S and Axis 1 is negative in L. donovani
and L. major but positive in L. infantum. Conversely,
the correlation between NC and Axis 1 is positive in L.
donovani and L. major but negative in L. infantum.
The correlations between GC3S and Axis 1 suggests
that highly biased genes, those with G/C-ending
codons, are clustered on the negative side in L. dono-
vani and L. major but on the positive side of Axis 1 in
L. infantum (Table 1). Also, the high degree of corre-
lation between GC3S and Axis 1 suggests that direc-
tional mutational pressure plays a major role in gov-
erning the synonymous codon usage. In addition, the
low value of NC (Table 1) indicates that HEGs are un-
der translational selection. In L. donovani, GT3S,
gravy and aromaticity all significantly contributed to
the variation on Axis 2. In L. infantum, both GT3S and
gravy significantly correlated with Axis 2 in L. donovani,

Table 1 Major trends in synonymous codon usage in L. donovani, L. infantum and L. major as revealed by COA on RSCU of
genes on Axis 1 and 2
Axis 1
Axis 2
Organism
Total variability Source of variation
Correlation
coefficient (r)a
Total variability
Source of
variation
Correlation
coefficient (r)a
31.5% NC 0.693
0.983
7.4% GT3S
0.371
GC3S Aromaticity
0.358
L. donovani



Gravy
0.265
15.7% NC
0.940
4.7% GT3S 0.621
L. infantum


GC3S 0.951 Gravy
0.131
17.2% NC 0.957 4.7% Aromaticity
0.117 L. major


GC3S
0.953

Note: aAll correlations are significant at P<0.01.

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
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while in L. major, aromaticity was found to be the
only major source of variation on Axis 2.
A plot of Axis 1–Axis 2 of each genome under
study including L. donovani, L. major, and L. infan-
tum was drawn, showing that HEGs are clustered at
one end of Axis 1 (Figure 1, circle), indicating that
these genes follow a distinct pattern of synonymous
codon usage.
A comparison of RSCU values of the HEGs with
those of the LEGs shows that in all three parasites
examined, a similar subset of synonymous codons,
mostly G/C-ending, are preferred by the HEGs (Table
S1, codons with bold values). The LEGs exhibit rela
tively higher usage of A/U-ending codons. But in all
three species, even the LEGs prefer to use
G/C-endingcodons for most of the amino acids,
though the frquencies of such codons are low. This is
in agreement with the high GC content in the genes
from L. donovani (58.8%), L. infantum (59.3%) and L.
major (59.7%). As seen in Table S1, high extent of
bias in the synonymous codon usage suggests that the
influence of translational selection is strong in all the
three Leishmania species.
Codon usage in variant surface glycoproteins,
HEGs and the topoisomerase gene
Variable surface glycoproteins (VSGs) have been
identified as parasite virulence factors that make pos-
sible the survival of Leishmania inside the macro
phages (15). DNA topoisomerases are a family of
DNA-processing enzymes involved in catalysis of the
breakage and rejoining of DNA strands (16). DNA
topoisomerase of L. donovani is distinct from other
eukaryotic counterparts with respect to its biological
properties and preferential sensitivity to many

Figure 1 Position of genes along the two major axes of
variation in COA on RSCU. Position of genes along Axis
1 are plotted against Axis 2 by COA in L. donovani (A),
L. infantum (B), and L. major (C). HEG, VSG, and
topoisomerase is represented by open circle, square and
triangle, respectively. The rest of the genes (LEGs) are
indicated by solid triangles. X and Y axis is represented
by Axis 1 and Axis 2, respectively. COA: correspondence
analysis; HEG: highly expressed gene; VSG: variable
surface glycoprotein; LEGs: lowly expressed genes.

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
Genomics Proteomics Bioinformatics 2011 Dec; 9(6): 218-228 221
therapeutic agents (17). Due to the therapeutical im-
portance of VSGs and topoisomerases, we have in-
cluded them separately for analysis of codon and amino
acid usage.
In all the three species of Leishmania examined,
genes other than HEGs constitute a single cluster
(Figure 1). But this is not the case for some genes, i.e.,
VSG and topoisomerase genes. Their highly scattered
nature on Axis 1–Axis 2 plot suggests that these
genes have different codon usage due to mutational
pressure or different translational selection. As indi-
cated in Figure 1 and Figure 2, all these genes are
also characterized by high GC3S and high NC values.
Major sources of variation in amino acid usages
To identify the major trends of intra-proteomic
variations in amino acid composition in the three
Leishhmania species, COA on amino acid usage was
performed. The first axis generated by COA accounts
for 32%, 24% and 30% of the total variations in L.
donovani, L. infantum, and L. major, respectively
(Table 2).
In all three species, codon adaptation index (CAI)
and GC12 were common primary sources of in-
tra-proteomic variations in amino acid usage (Table 2).
It was also observed that GC3S and NC provide addi-
tional trends of variability in all three Leishhmania
species. GT3S accounted for the variation on Axis 1 only
in L. donovani. Variation on Axis 2 was determined by
gravy and aromaticity for all three species. Observations
from Axis 1–Axis 2 plots of COA on amino acid usage
(Figure 3) showed that distribution of the HEGs in L.
donovani (Figure 3A) overlapped with that of other
genes. In L. infantum most of these genes lie on the left
side of Axis 1 (Figure 3B). In the case of L. major,

Figure 2 The NC plot of genes in the three Leishmania
species. NC is plotted against GC3S for L. donovani (A), L.
infantum (B) and L. major genes (C). The solid line indi-
cates the expected NC value if bias is due to GC3S alone.
HEG, VSG and topoisomerase is represented by open
circle, square and triangle, respectively. The rest of the
genes (LEGs) are indicated by solid triangles.

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
Genomics Proteomics Bioinformatics 2011 Dec; 9(6): 218-228 222
Table 2 Major trends in synonymous codon usage in L. donovani, L. infantum and L. major as revealed by COA on amino
acid usage of encoded proteins on Axis 1 and Axis 2
Axis 1 Axis 2
Organism
Total variability
Source of
variation
Correlation
coefficient (r)a
Total variability
Source of
variation
Correlation
coefficient (r)a
L. donovani 32% CAI
0.599
20% Aromaticity
0.792
NC 0.540 Gravy
0.717
GT3S
0.541

GC12 0.767
L. infantum 24.2% CAI
0.645
14.3% Aromaticity 0.397
NC 0.430 Gravy 0.334
GC3S
0.424

GC12 0.946
L. major 30% CAI 0.529 14.3% Aromaticity 0.669
GC3S 0.400 Gravy 0.531
GC12
0.862

Note: aAll correlations are significant at P=0.01. GC12: G/C content at first and second codon sites; CAI, codon adaptation index.

Figure 3 Position of genes along the two major axes of
variation in COA on RAAU. Position of genes along Axis
1 are plotted against Axis 2 by COA on RAAU in L.
donovani (A), L. infantum (B) and L. major (C). HEG,
VSG, and topoisomerase is represented by open circle,
square and triangle, respectively. The rest of the genes
(LEGs) are indicated by solid triangles. X and Y axis is
represented by Axis 1 and Axis 2, respectively.

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
Genomics Proteomics Bioinformatics 2011 Dec; 9(6): 218-228 223
HEGs clustered at the right side of the Axis 1 (Figure
3C). Figure 2 (A and B) and Table 2 together suggest
that the HEGs of L. donovani and L. infantum are char-
acterized by relatively high GC12. However, GC12 was
low in L. major, which was not expected because of the
high GC content in L. major. This may be due to the
effect of mutational pressure on L. major.
GC1 (G/C content at first codon sites) and GC2
(G/C content at first codon sites) of HEGs are similar
in all three species (Table S2). GC1 and GC2 of HEGs
in L. donovani are lower than those in LEGs in all
species, which could be due to mutational bias in L.
donovani, suggesting the higher AT content in LEGs
in L. donovani. Figure 4 shows the average amino
acid frequencies in proteins encoded by the HEGs and
LEGs in the three parasites under study. The fre-
quency of many amino acids differs in these two sets
of genes in L. major and is distributed widely,
whereas GC-rich codons are dominant in HEGs as
compared to LEGs (Figure 4, open and solid circles).
But this distribution is restricted to one extreme end in
the case of L. donovani (Figure 4, open and solid stars)
and L. infantum (Figure 4, open and solid squares).
These data suggest that there is a major variation in
selecting the codons for amino acids usage.

Figure 4 Frequency of amino acid residues in proteins en-
coded by HEGs and LEGs in three Leishmania species. Open
stars, squares and circles represent HEGs in L. donovani, L.
infantum and L. major, respectively and solid stars, squares and
circles indicate LEGs in L. donovani, L. infantum and L. major,
respectively. The amino acid (single letter code) residues are
showed on X axis and the frequency of amino acid occurring in
proteins encoded by LEGs and HEGs is indicated on Y axis.
Conservation of HEGs
Estimation of dS (number of synonymous substitu-
tions per synonymous sites) and dN (non-synonymous
substitutions per non-synonymous site) on the
orthologs of HEGs in L. donovani with L. infantum
and L. major was performed to investigate the evolu-
tion of amino acid substitution. Pairwise alignment
was done between the orthologs of HEGs of L. dono-
vani–L. infantum and L. donovani–L. major, and the
total numbers of synonymous substitutions and non-
synonymous substitutions are calculated. Table 3
shows that dN is higher than dS in both the groups. It
is noteworthy that the dS and dN values of L. dono-
vani–L. major are lower, while the dN/dS ratio is
higher than those of L. donovani–L. infantum. This
means that L. infantum has deviated at the synony-
mous and non-synonymous codon positions at a much
faster rate than L. major.
Codon and amino acid usage analysis for ho-
mologous genes
According to COA on RSCU, Axis 1 accounts for
30.54%, 26.47% and 32.53% of the total variations
due to GC3S and NC in three species (Table 4). On
Axis 2, GT3S and aromaticity account for the major
trends of variation. NC is correlated with Axis 1 posi-
tively in L. infantum but negatively in L. donovani
and L. major, while an opposite trend was observed
for the correlation between GC3S and Axis 1 in these
three species, suggesting that the genes with G/C-
ending codons are clustered on the right side but on
the negative side in L. infantum due to negative cor-
relation (Figure 5, Axis 1–Axis 2 plot of homologous
genes). It has also been noted (Table 4) that NC is
negatively correlated with Axis 1 in L. donovani and L.
major, which may be due to the decrease in codon bias
among the genes lying towards the right side of Axis 1.
This high correlation suggests that directional muta-
tional pressure is dominating for governing synony-
mous codon usage.
Table 3 dS and dN in orthologs of HEGs
Ortholog pairs dS dN dN /dS
L. donovani–L. infantum 0.094 0.12 1.27
L. donovani–L. major 0.056 0.074 1.32

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
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Table 4 Major trends in synonymous codon usage of homologous genes in three Leishmania species as revealed by COA on
codon usage of encoded proteins on Axis 1 and 2
Axis 1 Axis 2
Organism
Total variability
Source of
variation
Correlation
coefficient (r)a
Total variability
Source of
variation
Correlation
coefficient (r)a
L. donovani 30.54% NC
0.797
7.74% GT3S 0.338
GC3S 0.982 Aromaticity 0.125
L. infantum 26.47% NC 0.891 8.13% GT3S 0.237
GC3S
0.958
Aromaticity
0.309
Gravy
0.408
L. major 32.53% NC
0.761
6.95% GT3S
0.223
GC3S 0.976 Aromaticity
0.288
Gravy
0.221
Note: aAll correlations are significant at P<0.01.

Figure 5 Position of homologous genes along the two
major axes of variation in COA on RSCU. Position of
homologous genes along Axis 1 were plotted against Axis 2
by COA on RSCU in L. donovani (A), L. infantum (B), and
L. major (C). HEGs, VSG, and topoisomerase are
represented by circle, square, and triangle, respectively. The
rest of the genes (LEGs) are indicated by solid triangles.

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
Genomics Proteomics Bioinformatics 2011 Dec; 9(6): 218-228 225
NC–GC3S plot (Figure 6) indicates that HEGs consti-
tute a single cluster, but VSG and topoisomerase
genes demonstrate different codon usage pattern and
are characterized by high NC and GC3S for L. infantum
and L. major, while topoisomerase genes in L. dono-
vani are distributed randomly (range 0.42- 0.86).
COA on amino acid usage has been performed for
teomic variability. Axis 1 accounts for 33.51%, 39.3%
and 31.59% of total variation in the three species of
Leishmania (Table 5). CAI is the common source of
intra-proteomic variation in all species. GC12 accounts
for the additional variation in L. donovani and L. in-
fantum, while for L. major, gravy and aromaticity
contribute to variation besides CAI. Variation on Axis
2 is determined by gravy and aromaticity in L. dono-
vani and L. infantum, while for L. major, GC12 and
GT3S were the main contributors for the in-
tra-proteomic variation on Axis 2. In all three species,
HEGs, when plotted on Axis 1–Axis 2 (Figure S1),
were scattered, which was not expected because the
average GC content of these species is high. This dis-
crepancy may be due to the influence of mutational
pressure.
Discussion
The present study reveals the major trends involved in
the selection of gene/protein composition of the three
Leishmania species examined. The analysis of syn-
onymous codon usage and amino acid variations shows
that genomes of all the three Leishmania species are
under mutational bias and translational selection.
In all three species, the lower GC12 and higher

Figure 6 The NC plot of homologous genes in the three
Leishmania species. NC is plotted against GC3S for L.
donovani (A), L. infantum (B) and L. major genes (C). The
solid line indicates the expected NC value if bias is due to
GC3S alone. HEG, VSG, and topoisomerase is represented
by open circle, square and triangle, respectively. The rest
of the genes (LEGs) are indicated by solid triangles.

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
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Table 5 Major trends in synonymous codon usage of homologous genes in 3 Leishmania species as revealed by COA on
amino acid usage of encoded proteins on Axis 1 and 2
Axis 1 Axis 2
Organism
Total variability
Source of
variation
Correlation
coefficient (r)a
Total variability
Source of
variation
Correlation
coefficient (r)a
L. donovani 33.51% CAI 0.569 19.51% Gravy 0.629
GC12
0.761
Aromaticity 0.751
GT3S 0.519
L. infantum 39.3% CAI 0.478 8.13% Aromaticity 0.667
GC12
0.557
Gravy 0.550
L. major 31.59% CAI 0.443 19.5% GT3S
0.460
Gravy 0.567 Aromaticity
0.242
Aromaticity 0.700 GC12 0.715

Note: aAll correlations are significant at P ≤ 0.01.

GC3S in HEGs as compared to LEGs suggest that the
ancestor of the HEGs might have been relatively rich
in AT-content. Previous studies have suggested a
universal AT mutational bias, because many types of
spontaneous mutations (e.g., the deamination of cy-
tosine) cause GC to AT changes (18). This also sug-
gests that the LEGs have evolved at a faster rate and
become GC-rich. The lower dS and dN values in L.
donovani–L. major than those in L. donovani–L. in-
fantum suggests that the HEG dataset of L. donovani
is evolutionarily much closer to L. major. The higher
value of dN as compared to dS shows that synony-
mous positions are more conserved between L.
donovani and L. major, and mutational bias plays a
major role in shaping the composition of pro-
tein-coding genes. Additionally, optimal codons in
all three Leishmania species are G/C-ending in
HEGs but A/T-ending in LEGs. This supports the
fact that translational selection works more strongly
on synonymous sites of HEGs (19-21). As a result,
the HEGs of these three species are characterized by
low GC12 and high GC3S in comparison to the LEGs.
The HEGs may further be explored to identify the
essential genes, for example, by applying in silico
subtracting genomic approach, and could be helpful
in searching potential therapeutic drug targets for
curing leishmaniasis.
Materials and Methods
Sequence dataset
Complete protein-coding gene sequences of L. infan-
tum and L. major were extracted from the Sanger da-
tabase (http://www.sanger.ac.uk/)
tein-coding sequences of L. donovani were obtained
from NCBI (http://www.ncbi.nlm.nih.gov/), which
contain 2,655, 9,159 and 368 (till April 30, 2011)
protein-coding genes, respectively. To minimize sam-
pling error, genes with less than 100 codons, internal
stop codons, not-translatable codons, incomplete start
and stop codons, and pseudogenes were excluded
from the analysis. Therefore, finally 2,559 and 8,132
genes were included for analysis for L. infantum and
L. major, respectively. No such filter was applied for
L. donovani due to fewer gene sequences.
Homologs for L. donovani were searched using
BLAST. For this purpose the E-value cut-off was set
to e-100 and genes with E-value less than e-100 were
considered as homologs. According to this criterion, a
total of 341 genes from L. infantum and 340 genes
from L. major were found as homologs for 347 genes
from L. donovani.
while pro-

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Chauhan et al. / Codon and Amino Acid Usage Analysis in Leishmania
Genomics Proteomics Bioinformatics 2011 Dec; 9(6): 218-228 227
Parameters used for identifying trends of
variations
For each protein-coding gene under study, the fol-
lowing parameters were calculated, which include
RSCU, RAAU, CAI, GC12, GC3S at synonymous
codon sites excluding ATG for Met, TGG for Trp and
stop codons, average hydropathy (22) and aromaticity
(23) of the gene products.
Datasets of HEGs and LEGs
Datasets of putative HEGs and LEGs were obtained
by taking genes from the two extreme ends of Axis 1
of COA on RSCU in all three parasites.
Statistical analyses
The program CodonW 1.1.4 (Peden, J., 1999. avail-
able at http://sourceforge.net/projects/codonw/) was
used to analyze codon usage, COA (24), GC3S, RSCU
(22), and CAI (14, 18). A 2×2 contingency table χ2
was used to detect the significant differences in codon
and amino acid usage.
Estimation of non-synonymous and synony-
mous substitutions in HEGs
Orthologs for HEGs (genes lying at the one extreme
end of Axis 1 of COA) of L. donovani were extracted
using BLAST. The cut-off E-value for searching
orthologs was set to e-50 so the homologs with
E-value less than e-50 were considered as orthologs.
Pairwise alignments between the orthologs and esti-
mation of dS and dN were carried out using MEGA4
program (25).
Acknowledgements
The authors are thankful to the Sub-Distributed Informa-
tion Center (BTISnet SubDIC) and Department of Bio-
technology, BIT, Mesra, Ranchi for their kind support.
Authors’ contributions
NC and RP were involved in this study on all aspects,
contributed to the design of the project and wrote the
manuscript. ASV performed
non-synonymous substitutions analysis. All authors
read and approved the final manuscript.
synonymous/
Competing interests
The authors have declared that no competing interests
exist.
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Supplementary Material
Tables S1and S2; Figure S1
DOI: 10.1016/S1672-0229(11)60025-9