Immunogenetics (2010) 62:123–136
DOI 10.1007/s00251-009-0421-8

ORIGINAL PAPER

Study of cynomolgus monkey (Macaca fascicularis) DRA
polymorphism in four populations
Alice Aarnink & Laurent Estrade & Pol-André Apoil &Yuki F. Kita & Naruya Saitou & Takashi Shiina &Antoine Blancher

Received: 17 October 2009 / Accepted: 21 December 2009 / Published online: 22 January 2010
# Springer-Verlag 2010

Abstract To describe the polymorphism of the DRA genein Macaca fascicularis, we have studied 141 animals either
at cDNA level (78 animals from Mauritius, the Philippines,
and Vietnam) or genomic level (63 animals from thePhilippines, Indonesia, and Vietnam). In total, we characterized 22 cDNA DRA alleles, 13 of which had not been
described until now. In the Mauritius population, we
confirmed the presence of three DRA alleles. In thePhilippine and Vietnam populations, we observed 11 and
14 DRA alleles, respectively. Only two alleles were present
in all three populations. All DRA alleles but one differ fromthe consensus sequence by one to three mutations, mostbeing synonymous; so, only seven DR alpha proteins werededuced from the 22 cDNA alleles. One DRA cDNA allele,
Mafa-DRA*02010101, differs from all other alleles by 11 to
14 mutations of which only four are non-synonymous. The
two amino acid changes inside the peptide groove of
Mafa-DRA*02010101 are highly conservative. The very
low proportion of non-synonymous/synonymous mutations
is compatible with a purifying selection which is comparable to all previous observations concerning the evolutionof the DRA gene in mammals. Homologues of the allele
Mafa-DRA*02010101 are also found in two other Asian
macaques (Macaca mulatta and Macaca nemestrina). The
forces able to maintain this highly divergent allele in threedifferent macaque species remain hypothetical.
Keywords MHC . DRA . Macaque . Polymorphism

Electronic supplementary material The online version of this article(doi:10.1007/s00251-009-0421-8) contains supplementary material,
which is available to authorized users.
A. Aarnink : L. Estrade : P.-A. Apoil : A. Blancher (*)
Laboratoire d’immunogénétique moléculaire, EA3034,
Faculté de Médecine Purpan, Toulouse 3, IFR150 (INSERM),
CHU de Toulouse, Université Paul Sabatier,
Laboratoire d’Immunologie, Hôpital Rangueil,
1 Avenue Jean Poulhes, TSA 50032,
31059 Toulouse Cedex 9, France
e-mail: blancher.a@chu-toulouse.fr
Y. F. Kita : T. Shiina
Department of Molecular Life Science, Division of Basic
Medical Science and Molecular Medicine, Tokai University
School of Medicine,
143 Shimokasuya,
Isehara, Kanagawa 259-1143, Japan
N. Saitou
Division of Population Genetics, National Institute of Genetics,
Mishima 411-8540, Japan

Introduction
The cynomolgus macaque (Macaca fascicularis) is increasingly used as a nonhuman primate model for biomedical
research in various domains such as tissue allograft
rejection (Aoyama et al. 2009; Borie et al. 2002; Wieczorek
et al. 2006), bone marrow graft (Derdouch et al. 2008; Lauet al. 2004), immune response against pathogens (Sato et al.
2008), or new vaccines (Kita et al. 2005; Morner et al.
2009; Turbant et al. 2009; Warfield et al. 2007). Recently,
its use in simian immunodeficiency virus-induced AIDS
(Burwitz et al. 2009; Florese et al. 2008; Mee et al. 2009b)
or with other emergent pathogens (Baskin et al. 2009;
Kuiken et al. 2003; Lawler et al. 2006; Reed et al. 2005)
revealed the importance of the knowledge of the genetic
background. Moreover, it was reported that the geographical origin of the animal can influence the response to drugs

124

or sensibility to the experimental disease (Menninger et al.
2002; Schmidt et al. 1977). The polymorphism of some lociinvolved in the physiology of the immune system has been
extensively studied such as DRB (Blancher et al. 2006;
Leuchte et al. 2004) MHC class I (Campbell et al. 2009;
Pendley et al. 2008; Uda et al. 2005; Uda et al. 2004), theimmunoglobulin locus (Calvas et al. 1999), the KIR locus(Bimber et al. 2008), chemokine and chemokine receptors(Puissant et al. 2003).
The map of the cynomolgus macaque MHC has
recently been characterized (Watanabe et al. 2007). It
demonstrates that the cynomolgus MHC shares the generalorganization of the rhesus monkey MHC characterized bynumerous class I duplicated genes (Daza-Vamenta et al.
2004). This contrasts with the high homology in the class
II region with humans as well as other primates. In theclass II region, DR, DQ, and DP gene polymorphisms
have been extensively characterized in M. fascicularis(Blancher et al. 2006; Leuchte et al. 2004; Otting et al.
2002; Sano et al. 2006). By contrast, the polymorphism of
the DRA gene was studied only in the Mafa Mauritius
population (O'Connor et al. 2007) which is well known tohave a highly reduced polymorphism due to a founding
effect (Blancher et al. 2008; Bonhomme et al. 2008;
Lawler et al. 1995). We decided to study the DRA
polymorphism in the coding region by the study of cDNAin animals from three of the four populations we havealready studied (from Mauritius, the Philippines, andVietnam). Vietnam was chosen as an example of thecontinental subpopulation. The Mauritius population was
included because of its interest in preclinical models(Florese et al. 2008; Mee et al. 2009b). The Philippine
population was studied because, as we have previously
reported, this population probably experienced a strong
selection in the class II region of the MHC (Bonhomme etal. 2007).

Materials and methods
Animals
In total, we studied 298 unrelated cynomolgus monkeys
on Mauritius island (samples provided by NoveprimCo.), 212 from the Philippine archipelago (200 from
Sicombrec Co. and 12 from INA Research Philippines
INC), 88 from Vietnam (83 from Nafovanny Co. andfive from the Shiga University of Medical Science,
Shiga, Japan), and 13 from Indonesia (CV. Universal
Fauna Breeder). Two unrelated Macaca sylvanus (Masy)(Ecole Nationale Vétérinaire, Toulouse, France) and one
Macaca nesmestrina (Mane) (Ecole Nationale Vétérinaire, Toulouse, France) were studied. The blood collection

Immunogenetics (2010) 62:123–136

and animal studies were conducted in accordance with theguidelines for animal experiments specific to eachlocation.
DNA extraction
Genomic DNA was extracted from peripheral blood using
either QIA amp Blood Kit (Qiagen, Courtaboeuf, France)or a standard phenol–chloroform method.
RNA extraction
Total RNA was extracted from buffy-coats by means of an
RNeasy Mini Kit (Qiagen, Courtaboeuf, France).
Microsatellite analysis
In the three populations (on Mauritius island, the Philippines, and Vietnam), we studied DRACA microsatellite, aspreviously described (Bonhomme et al. 2005). For the
Mauritius population, we have also studied microsatellites
located in the MHC class II region (D6S2892, D6S2876,
D6S2747, D6S2745, and D6S2771) by means of primers
described by Wiseman et al. (2007). Genotypes were
determined using DNA Size Standard-kit-600 (Beckman
Coulter, Villepinte, France) after denaturation and separation of the amplification products on a capillary electrophoresis using CEQ8000 analyzer and scored with the software
CEQ8000 Genetic Analysis System v8.0 (Beckman
Coulter, Villepinte, France).
Genetic diversity
Genetic diversity was measured using the number ofalleles (nA) and the expected and observed heterozygosity(He and Ho), calculated with Arlequin v.3.1 software(Excoffier et al. 2005). To test departures from Hardy–
Weinberg equilibrium, the Fis statistics within samples, andthe corresponding exact P values were computed by aMarkov chain method implemented in Genetix v4.05.2(Belkhir et al. 2004). The linkage disequilibrium (LD)between the microsatellite DRACA, DRA and the DRB
region was tested using ratio test, implemented in Arlequinv.3.1 which gives the significance level of LD by thecalculation of an exact P value obtained from multiple
permutations.
Characterization of DRB alleles by denaturing gradient gelelectrophoresis and sequencing
Genomic DRB exon 2 sequences of animals from Mauritius(N=298), the Philippines (N=200), and Vietnam (N=50)
were determined by denaturing gradient gel electrophoresis

Immunogenetics (2010) 62:123–136

and sequencing (DGGE-sequencing) as described previously (Blancher et al. 2006).
Characterization of DRA cDNA sequences (cDRA)A DRA cDNA fragment of 734 base pairs (bp) was
obtained by using the one-step reverse transcriptase (RT)PCR kit from Qiagen (Qiagen, Courtaboeuf, France), withprimers DRAmac1Forward 5′-ATG GCC GAA AGT GGA
GTC C-3′and DRAmac774Reverse 5′-GCA TAT GTC TCACAG AGG CCC-3′. The cycling parameters were 30 min at
50°C to obtain cDNA (reverse transcription reaction anddenaturation of the cDNA template), followed by 15 min at
95°C (activation of Hot Star Taq polymerase and inactivation of reverse transcriptase), followed by 35 cycles of 30 s
at 94°C denaturation step, an annealing step of 30 s at
60°C, an extension step of 1 min at 72°C, and 10 min at
72°C (final extension). When compared with the full-length
coding region, the resulting amplified fragments lack 19
and 12 nucleotides at the beginning and the end, respectively. The resulting amplified products were separated on
agarose gels, purified using QIAquick Gel Extraction Kit(Qiagen, Courtaboeuf, France) and directly sequenced on
both strands by using both DRAmac1Forward, DRAmac
774Reverse, DRAmac421Forward 5′-GTG GTC AAA
GTT ACA TGG C-3′, and DRAmac475Reverse 5′-CTG
ACA CTC CTG TGG TGA C-3′ primers with thefluorescent Dye Terminator Cycle Sequencing method on
a CEQ 8000 automated sequencer (Beckman Coulter,
Villepinte, France). When direct sequencing revealed noambiguities, the animal was considered to be homozygous
and the sequence was validated after having been found in
at least two unrelated animals. When direct sequencing
revealed ambiguities, the DRA cDNA amplified fragments
were cloned into the pCR2.1 Topo plasmid vector (Invitrogen, Leek, The Netherlands) and sequenced to allow
characterization of both DRA cDNA alleles.
Characterization of DRA gDNA sequences
A DRA DNA fragment of 1,198 to 1,318 bp from partial
intron 1 to partial intron 3 was obtained by PCR
amplification, using Gold Taq polymerase (ABI), withsense primers DRA-F1 5′-AGCATGTCCTTCACCCAGA
ACT-3′ or DRA-F2 5′-ACTTCCTGCCTACATGTATG-3′,
and the anti-sense primers DRA-R1 5′-TAAAGGAGATTGAGTGATGGTCCA-3′ or DRA-R2 5′-AAGCTGGGTAGTAGGAGAAG-3′. The cycling parameters were 10 min at
95°C, followed by 30 cycles of 30 s at 94°C denaturation
step, an annealing step of 20 s at 65°C, and an extension
step of 2 min at 72°C and 5 min at 72°C (final extension).
PCR products were cloned into the PGEM-T Easy vector
with the TA cloning kit according to the protocol provided

125

by the manufacturer (Promega, Madison, WI) and sequenced by using the ABI3130 genetic analyzer (Applied
Biosystems, CA) in accordance with the protocol of the Big
Dye terminator method. To avoid PCR and sequencing
artifacts generated by polymerase errors, 16 clones weresequenced per individual. The nucleotide sequences of all
individuals were also determined by direct sequencing of
the PCR products using PCR primers as sequencing
primers.
Sequence analysis and phylogenic trees
All DRA sequences were deposited in the Genbank
database (accession references are given in the tables) andsubmitted to the IPD-MHC NHP database (Robinson et al.
2005) for allele nomenclature. Cynomolgus monkey allele
names were assigned to DRA sequences by the IPD-MHC
NPH database following classical rules (Klein et al. 1990).
Multiple sequence alignments were obtained by using
ClustalW 1.83 (Thompson et al. 1994). Phylogenetic
analyses were conducted in MEGA4 (Tamura et al. 2007).
The phylogenetic trees were built using the neighborjoining method (Saitou and Nei 1987) with evolutionary
distances computed by using the Maximum Composite
Likelihood method (Tamura et al. 2004). All positions
containing gaps and missing data were eliminated from thedataset (complete deletion option). A phylogenetic network
was built to describe all possible phylogenetic pathways of
the DRA coding sequences of Mafa, Mamu (Macaca
mulatta), Mane, and Masy. The network was rooted byusing the human HLA–DRA sequence as outgroup.
Study of EST databases
We extracted 154 DRA expressed sequence tag (EST) fromthree libraries (thymus [QthA, Lib.24021, n=9,566], spleen[QspA, Lib.23009, n=9,783] and bone marrow [QbmA,
Lib.22708, n=9472], available in the Unigene database(http://www.ncbi.nlm.nih.gov/UniGene/lbrowse2.cgi),
obtained from a 4-year-old male Malaysian M. fascicularis(Osada et al. 2009). For each library, we built DRA cDNA
consensus sequence by using Macvector software (Macvector
Inc, Cary, NC, USA).

Results
Polymorphism of microsatellite DRACA and linkage
disequilibrium
We studied the polymorphism of the DRA-associated
DRACA microsatellite in three cynomolgus macaquepopulations from Mauritius island (N=298, Noveprim),

126

the Philippines archipelago (N=200, Sicombrec), andVietnam (N = 50, Nafovanny). All the animals were
genotyped for the DRB genes by DGGE and sequencing,
and the DRB haplotypes were deduced from the Mauritius
and the Philippine reference haplotypes already described(Blancher et al. 2006). In the Vietnamese population, thehigh degree of polymorphism of the DRB region did notallow us to deduce the DRB haplotypes (to be publishedelsewhere).
In total, we found 21 alleles of microsatellite DRACA
among which only three are in common in the three
populations (Table 1). The Mauritius sample is the least
polymorphic with only five alleles which are common with
the Vietnamese sample. The latter is the most polymorphic
with 17 alleles, eight of which are common with thePhilippines sample, in which we found 12 alleles.
Statistical tests did not reveal Hardy–Weinberg equilibrium departure in the Mauritius or the Philippine samples.
In contrast, in the Vietnamese population sample, we
observed an excess of homozygous animals (Table 2).
The linkage disequilibrium between DRACA and DRB
haplotypes is complete in the Mauritius population sample(p<10-6). The DRACA–DRB haplotypes are shown in
Fig. 1. In the Philippines population sample, the DRACA–DRB LD was almost complete (p<10-6), and the mostfrequent haplotypes are shown in Fig. 2 which also describes
one recombinant DRACA–DRB haplotype.
There is no direct, specific concordance between microsatellite allele sizes and cDNA allele sequences. For
instance, in the Mauritian cynomolgus macaques, there
are five microsatellite allele sizes but only three cDNA
allele sequences (Fig. 1). In the Mauritius population, theLD between DRACA microsatellite and cDRA allele
cannot be calculated on the basis of the eight animals
studied here. The compilation of our results with those from
O'Connor is not possible because insufficient details areprovided in the publication from this group. In thePhilippine population, the LD between DRACA microsatellite and cDRA is highly probable (p<10-6; N=20
animals). In the Vietnamese population, on the basis on
the 50 animals studied here, the LD between the DRACA
microsatellite and cDRA is highly probable (p< 10-6)although the description of Vietnamese DRACA–DRA
haplotypes remains elusive.
Study of cDNA DRA sequences
We studied 78 unrelated cynomolgus monkeys from threedifferent populations: Mauritius island (N=8, Noveprim),
the Philippines archipelago (N = 20, Sicombrec), andVietnam (N=50, Nafovanny). In the Mauritius and Philippine populations, the animals were selected for their DRB
genotype with the aim to explore the DRA alleles

Immunogenetics (2010) 62:123–136

associated to each DRACA–DRB haplotype of both populations. For the most frequent haplotypes, we studied one
homozygous animal, while for the least frequent haplotypes, we studied heterozygous animals having one of therare haplotypes and one of the most frequent haplotypes. In
total, we deduced the cDNA DRA sequences associatedwith the most frequent and the rarest DRACA–DRB
haplotypes.
In the Mauritius population, seven frequent DRB
haplotypes are found (Blancher et al. 2006; Leuchte et al.
2004; O'Connor et al. 2007). Two rare recombinant DRB
haplotypes (#4c and #4d) were also described (Blancher et
al. 2006). The cDRA alleles associated with these two rarerecombinant haplotypes were not studied because no RNA
samples from appropriate animals were available. In total,
we describe here seven DRACA–DRA–DRB haplotypes
deduced from the study of eight animals by cloning andsequencing of DRA cDNA (Fig. 1). We confirm the results
reported by O'Connor et al. for six DRACA–DRA–DRB
haplotypes (O'Connor et al. 2007), and we report here thefirst the association of Mafa–DRA*01020101 with the
haplotype DRB #3 (Leuchte et al. 2004; Blancher et al.
2006) and the microsatellite-defined haplotype H7 (Mee etal. 2009a; see Fig. 1). In total, in the Mauritius sample,
only three DRA cDNA alleles, previously described by
O’Connor and colleagues, are observed (Table 3; O'Connor
et al. 2007), out of which (Mafa-DRA*01020101 and
Mafa-DRA*02010101) were also found in Vietnam and
the Philippine animals (Table 3), while the third (MafaDRA*010101) was found only in Mauritius monkeys(Table 3). Even by the study of genomic sequences, this
Mauritius allele was absent in the samples from Vietnam,
the Philippines, and Java (see further section).
In the Philippine population, twelve DRB haplotypes
have previously been described (Blancher et al. 2006). Twoof them (#1b and #13b) were not studied for the cDRAassociated sequences because no RNA samples wereavailable (see foot note for haplotype #1b1). We describe
here for the first time one more Philippine DRB haplotype,referred to as DRB #2b, which displays two DRB loci(Fig. 2b). At one locus, haplotype #2b shares the allele(DRB*W501) with the Mauritius DRB haplotype #2a,while at the second locus, the Philippine haplotype #2b
displays the allele DRB*W7201 (Genbank access number
FJ655856) which differs by six mutations in exon 2 fromallele DRB*W2101 found in the Mauritius DRB haplotype#2a. In total, we studied 20 animals here representative of1
Writing the present article, we detected an error concerning DRB
haplotype #1b that we reported in the article Blancher et al. 2006
(Fig. 2). The latter haplotype is absent from the Mauritius population
but was observed in a single animal from The Philippines, while
haplotype #1a was not observed in The Philippine sample. We
apologize for this error.

Immunogenetics (2010) 62:123–136
Table 1 Allelic frequencies(in %) of DRACA microsatellite
in three cynomolgus macaquepopulations

Legend: N is the haplotype
number studied for each
population

127
Microsatellite length (bp)

Mauritius (N=596)

The Philippines (N=400)

Vietnam (N=100)

237
239
241
247
249
251
253
255
257
259
261
265
266
268
270

24.8
0
0
0
0
0
0
0
0
0
0
0
21.1
29.0
2.0

19.5
42.3
0
0
0
0.5
0
0
7.8
0
0.5
12.5
0
0
0.5

1.0
5.0
3.0
8.0
11.0
0
4.0
3.0
2.0
1.0
0
6.0
2.0
5.0
13.0

272
274
276
278
283
286
allele number

23.0
0
0
0
0
0
5

0.8
5.5
5.3
0
4.5
0.5
12

27.0
4.0
1.0
4.0
0
0
17

11 Philippine DRB haplotypes and have evidenced 11
cDRA alleles (Table 3). Figure 2b describes the DRACA–
cDRA–DRB haplotypes deduced from the study of 20
animals (Fig. 2a). Two DRB haplotypes (#5 and #10) areassociated with several cDRA (Fig. 2) and symmetrically,
three out of 11 cDRA alleles (Mafa-DRA*01020101, MafaDRA*01020701, and Mafa-DRA*01010401) are associated with more than one DRB haplotype. Interestingly, oneclass II Philippine haplotype is compatible with a recombination between two other class II Philippine haplotypes(see Fig. 2b).
As for the Vietnamese population, because the great
diversity of the DRB region did not allow us to determine
the DRB haplotypes, we decided to study DRA cDNA of 50
randomly chosen unrelated animals. In this sample, we
found 14 cDRA sequences (Table 3). Only four of theseVietnamese cDRA alleles are found in the two otherpopulations (Table 3). The most frequent Vietnamese allele,

Mafa-DRA*01020101, occurred in 39 out of the 50 animals,
out of which 29 appeared homozygous by direct sequencingof their amplified DRA cDNA. We found here that theobserved frequencies of cDRA homozygous animals exceed
largely the theoretical frequency (Ho=0.38, He=0.53,
p<10-6). In this 50-animal Vietnamese sample, an excess ofhomozygous animals was also observed for DRACA(Table 2). These observations led us to conclude that the
sample we studied most probably presented an excess ofhomozygous animals resulting from a sampling effect andthat the putative difficulty to reveal some heterozygous
animals by RT-PCR direct sequencing can reasonably beeliminated.
Comparison of DRA cDNA sequences
In total, we describe 22 DRA cDNA sequences in M.
fascicularis out of which four have already been described

Table 2 Genetic diversity at DRACA microsatellite in Mauritius, Philippine, and Vietnam populations
Population

nA

Ho

He

P valueFis

P value

Mauritius (298)the Philippines (200)Vietnam (50)

5
12
17

0.79
0.73
0.72

0.76
0.75
0.88

0.100
0.749
0.005

−0.04
0.03
0.19

0.08930.1844<0.0001

Legend: population (number of animals studied), nA number of alleles, He expected heterozygosity; Ho observed heterozygosity

128
Fig. 1 DRACA–DRA–DRB
haplotypes in Mauritius population. a For each animal studiedin Mauritius population (n=8),
we describe the DRACA microsatellite alleles, cDRA alleles,and DRB haplotypes. b Schematic representation of the
DRACA–DRA–DRB haplotype
in the Mauritius population (theorder of DRB genes on eachhaplotype is arbitrary). Haplotypes DRACA–DRA–DRB are
identified by numbers precededby “number sign” (these numbers correspond to the DRB haplotype numbers described
previously by Blancher et al.
2006) and by numbers following
the letter “H” corresponding to
the nomenclature of Wiseman etal. (2007). Animal identification
codes are given on the right ofeach haplotype

Immunogenetics (2010) 62:123–136aReference
animal

DRACA

cDRA

DRB
haplotype121518
42-23
OBDR5
OBDK5
13788
OBAN5
13744
OBCN5

272 / 272
237 / 237
270 / 270
266 / 266
266 / 266
268 / 272
268 / 268
268 / 268

DRA*01020101 / DRA*01020101
DRA*02010101 / DRA*02010101
DRA*01020101 / DRA*01020101
DRA*01020101 / DRA*01020101
DRA*010101 / DRA*010101
DRA*01020101 / DRA*01020101
DRA*01020101 / DRA*01020101
DRA*01020101 / DRA*01020101#1a / #1a
#2 / #2#3 / #3#4a / #4a#4b / #4b
#5 / #1a#5 / #6#6 / #6

DRA*01020101

DRB1*1002b

272

DRBW*4901

121518 ; OBAN5#1a – H3
237

DRA*02010101

270

DRA*01020101

DRB1*0302

266

DRA*01020101

DRB1*0401

266

DRA*010101

DRB*W501 DRB*W2101

42-23#2a – H1
DRB*W103

DRB*W3601#3 – H7#4a – H4

Microsatellite
haplotype inclass II
H3 / H3
H1 / H1
H7 / H7
H4 / H4
H5 / H5
H6 / H3
H6 / H2
H2 / H2OBDR5DRB4*0101 DRB5*0301

OBDK5
DRB4*0102 DRB5*0301

DRB1*0401

13788#4b – H5
268

DRA*01020101

268

DRA*01020101

DRB1*0402

DRB*W401

OBAN5 ; 13744#5 – H6#6 – H2

in M. fascicularis in Mauritius (O'Connor et al. 2007) andIndonesia (Mee E.T. and Rose N.J., unpublished data)(Table 3). One Mafa DRA cDNA sequence found in
Genbank (Mafa-DRA*01020301, AM943637), revealed inan Indonesian cynomolgus macaque, was not found in our
animal sample. The comparison of all Mafa cDRA aligned
sequences revealed 26 variable positions, 17 of which aresynonymous by reference to the consensus sequence which
is identical to the Mafa-DRA*01020101 allele (Fig. 3).
Among the nine variable amino acid positions, two arelocated in the leader peptide, three are in the alpha1
domain, one in the alpha2 domain, one in the connecting
peptide, and two in the trans-membrane domain (Fig. 3). By
and large, the dN/dS ratio (non-synonymous substitutions
per non-synonymous site divided by synonymous substitutions per synonymous site) was much lower than one in all
pairwise allele comparisons, suggesting a strong purifying
selection (Hughes and Nei 1989) (Electronic Supplementary Material 1).
All Mafa DRA cDNA sequences but one (MafaDRA*02010101) differ only by one to three mutations
from the consensus sequence (Fig. 3). The MafaDRA*02010101 sequence excluded, pairwise comparisonsof all other M. fascicularis sequences revealed from one tosix mutations with a ratio of non-synonymous/synonymous
mutations varying from 0/5 to 3/1 (see Fig. 3 for moredetails). The allele Mafa-DRA*02010101 which is present
in the three populations of M. fascicularis studied here
(Table 3), displays 11 to 14 mutations when compared toother Mafa-DRA alleles with a dN/dS ratio varying between
0.40 and 0.75. By comparison with other Mafa cDRA

DRB*W402

OBCN5 ; 13744

alleles, Mafa-DRA*02010101 displays eight specific mutations (not shared with any other allele). The deduced MafaDRA*02010101 protein differs from the six other deduced
Mafa DRA proteins by four specific (not shared with anyother allele) amino acid substitutions (Fig. 3). The
substitution in the leader peptide is not conservative (IleThr); the three substitutions located in the alpha 1 domain
are highly conservative (Glu-4-Asp, Phe-22-Tyr and Ile31-Leu, Fig. 3). Only two of them (Phe-22-Tyr and Ile-31Leu) involve amino acid residues in contact with theantigenic peptides presented by the DR groove (Recheand Reinherz 2003).
The comparison of the 22 Mafa cDNA sequences withhomologous sequences from other macaque species
revealed that eight of the Mafa cDRA sequences describedhere are identical to the DRA cDNA sequences described
previously in M. mulatta (Fig. 3 and Table 3). Among the
19 SNPs (single nucleotide polymorphisms) found in thecoding region of Mamu, almost all (17 out of 19) arepresent in M. fascicularis.
From the alignment of all the macaque DRA cDNA
sequences (Fig. 3), we produced a phylogenetic networkfrom the polymorphic sites of Mafa-, Mamu-, Masy-, ManeDRA cDNA alleles (Fig. 4). The network displays several
reticulations which suggest inter-allelic exchanges. In theneighbor-joining tree (Fig. 5), most of the macaque DRA
sequences form a star-like phylogeny which includes thetwo Masy alleles (Masy-DRA*01, EU877221 and MasyDRA*02, EU877222) described for the first time. A second
group including Mafa-DRA*02010101, Mamu-DRA*
010601 cDNA sequence and two Mane cDRA (see Figs. 5,

Immunogenetics (2010) 62:123–136
Reference
animalCyn-239
Cyn-314
Cyn-321
Cyn-355
Cyn-376
Cyn-381Cyn-388
Cyn-391
Cyn-475
Cyn-483
Cyn-491
Cyn-600
Cyn-610Cyn-611
Cyn-633
Cyn-695
Cyn-697
Cyn-705
Cyn-721
Cyn-810

b
#2b

#5a
#5b
#7
#8
#9
#10a
#10b
Rec [#9/#10]
#11
#12
#13

251

129
DRACA

cDRA

239 / 239
237 / 237
239 / 239
239 / 239
239 / 276
265 /265
251 / 239
239 / 286
237 / 272
257 / 261
265 /265
257 /257
274 / 265
237 / 265
239 / 272
239 / 274
276 / 265
239 / 283
251 / 237
251 / 257

DRA*010302 / DRA*010302
DRA*010301 / DRA*010301
DRA*010303 / DRA*010302
DRA*010303 / DRA*010303
DRA*010303 / DRA*0105
DRA*01010401 / DRA*01010401
DRA*02010101 / DRA*01020501
DRA*010303 / DRA*01020101
DRA*010301 / DRA*0109
DRA*01020701 / DRA*01020701
DRA*01010401 / DRA*01010401
DRA*01020701 / DRA*01020701
DRA*01020101 / DRA*01010401
DRA*010301 / DRA*01010401
DRA*010303 / DRA*0109
DRA*010303 / DRA*01020101
DRA*01040201 / DRA*01010401
DRA*010303 / DRA*01020201
DRA*02010101 / DRA*010301
DRA*02010101 / DRA*01020701

DRA*02010101#15 / #15
#11 / #11
#7 / #15#7 / #7#7 / #5#13a / #13a#2b / #7
#7 / #10#11 / #12
#9 / #9#13a / #13a
#9 / #9#8 / #14#11 / #14
#7 / #12#7 / #8#5 / #13a#7 / #10#2b / #11
#2b / #10

DRB*W501 DRB*W7201

Cyn-388 ; Cyn-721 ; Cyn-810
276

DRA*01040201

DRB1*0402

DRB*W401

276

DRA*0105

DRB1*0402

DRB*W401

239

DRA*010303

DRB1*0321 DRB1*1007

Cyn-697
Cyn-376
Cyn-355 ; Cyn-321 ; Cyn-376 ; Cyn-391 ;Cyn-705 ; Cyn-695 ; Cyn-633 ; Cyn-388

274

DRA*01020101

DRB1*0319

257*

DRA*01020701

DRB1*0307 DRB1*1006

DRB*W3301

283

DRA*01020201

DRB1*0318

DRB*W202

286

DRA*01020101

DRB1*0318

DRB*W202

257

DRA*01020701

DRB1*0318

DRB*W202

237

DRA*010301

DRB*W301 DRB*W3601

272

DRA*0109

DRB*W604

265

DRA*01010401

DRB*W603 DRB*W602 DRB3*402

265

DRA*01010401

239

DRA*010302

Cyn-695 ; Cyn-610
Cyn-483 ; Cyn-600
Cyn-705
Cyn-391
Cyn-810Cyn-314 ; Cyn-475 ; Cyn-721 ; Cyn-611
Cyn-633 ; Cyn-475 ;DRB1*0404

DRB*W3302#14#15

DRB haplotype

Cyn-381 ; Cyn-491 ; Cyn-697

DRB5*0302

Cyn-611 ; Cyn-610

DRB*W5301

Cyn-239 ; Cyn-321

Fig. 2 DRACA–DRA–DRB haplotypes in the Philippine population. a
For each animal studied in the Philippine population (n=20), we
describe the DRACA microsatellite alleles, cDRA alleles and DRB
haplotypes. b Schematic representation of the DRACA–DRA–DRB
haplotypes in the Philippine population (the order of DRB genes oneach haplotype is arbitrary). Haplotypes DRACA–DRA–DRB are
identified by numbers (given on the left) which correspond to the
DRB haplotype numbers described previously (Blancher et al. 2006).
Animal identification codes are given on the right of each haplotype.

Numbers with a ring around them correspond to DRACA–DRA–DRB
haplotypes found in homozygous animals. The other haplotypes werededuced by the study of heterozygous animals. As for haplotype #9,
the microsatellite length mostly encountered is 257, but one animal
homozygous for cDRA and haplotype DRB (Cyn-483) displayed two
lengths (257 and 261) for the DRACA microsatellite. All DRACA
alleles found in the sample of 200 animals described in Table 1, except
a rare one (DRACA allele 270 present only in two animals), belong to
the DRACA–DRA–DRB haplotypes defined here

3 for more details) is highly divergent. The allele MafaDRA*02010101 differs by 13 mutations from MafaDRA*01020101 which most probably corresponds to themacaque cDRA ancestral sequence (Fig. 4). This gives a 12million-year estimate for the time of divergence between
the two alleles (by calibration with the number of mutations
between human and macaque cDRA).

Study of DRA genomic DNA sequences
In order to enlarge our dataset, we also studied DRA partial
genomic sequences from animals for which no mRNA
samples were available. The animals studied were fromthree different populations [the Philippines (N=12, INA
Research Philippines INC), Indonesia (N=13, CV Univer-

130

Immunogenetics (2010) 62:123–136

Table 3 Cynomolgus monkey DRA allele distribution in the three populations studied and identical alleles described previously
IMGT/NPH Mafa-DRA allele names

Genbank
access codes

Presence of allele in populations
Mauritius

Philippines

Vietnam√

DRA*010101
DRA*01010401
DRA*010105
DRA*01020101

EU877215
EU877218
EU921295
EU877216

DRA*01020201
DRA*01020501
DRA*01020701
DRA*01020801
DRA*01020901
DRA*010212
DRA*010216
DRA*010218
DRA*010301

EU877210
EU877204
EU877217
EU877203
EU877205
EU877202
EU921296
EU921294
EU877212√

DRA*010302

EU877211√

DRA*010303
DRA*010304
DRA*010305
DRA*01040201
DRA*0105
DRA*0106

EU877209
EU877207
EU877206
EU877219
EU877214
EU877208√

DRA*0109
DRA*02010101

EU877213
EU877220

√
√

√

√
√

√
√
√
√
√
√
√
√
√

√
√
√
√
√
√

√
√

Previously described identical alleles,
Genbank access codes√√

Mafa-DRA*0101
nf
Mamu-DRA*010204
Mafa-DRA*01020101
Mamu-DRA*010201

EF208826(1)AJ586877(2)EF208827(1)AJ586874(2)

nf
nf
nf
nf
Mamu-DRA*010401
Mamu-DRA*010205
nf
Mamu-DRA*010207
Mafa-DRA*0103
Mamu-DRA*010301
Mane-DRA*01

AJ586880(2)AM943638(3)AJ586881(2)GQ214407(4)

nf
nf
nf
nf
nf
nf
Mamu-DRA*0105

AJ586884(2)nfMafa-DRA*02010101
Mamu-DRA*010601

EF208828(1)AM910165(5)AJ586882(2)AJ586878(2)aLegend:a

Expressed sequence tag (EST) of M. nemestrina available in Genbank (EB520956, EB521652) similar to Mafa-DRA*02010101. Reference
quoted in the tables: (1; O'Connor et al. 2007), (2; de Groot et al. 2004), (3; unpublished, deposited by Mee and Rose, found in Indonesiancynomolgus macaque), (4; an M. nemestrina animal, studied in this study, homozygous for an allele strictly identical with Mafa–DRA*010301),(5; full-length cDNA deduced from Mamu-DRA genomic DNA sequence published by Doxiadis et al. 2008), nf not foundsal Fauna Breeder), and Vietnam (N=38)]. From these 63
cynomolgus macaques, we found a total of 41 Mafa-DRA
genomic sequences (fragment of 1,198 to 1,318 base pairs
from partial intron 1 to partial intron 3) from which only 18
partial cDNA alleles (exons 2 and 3 only) are deduced. The
geographic origins of the animals that have led to thedescription of the alleles are given in Electronic Supplementary Material 2 where we also provide the Genbank
access codes and identity with cDRA allele describedabove. Among these 41 alleles, only two are in common
in the three populations (Mafa-DRA*01020101 and MafaDRA*01020401). The neighbor-joining phylogenetic treeof these genomic sequences (Electronic Supplementary
Material 4a) revealed that most describe a star phylogeny

while four sequences highly homologous to MafaDRA*02010101 are grouped on a highly divergent branch.
In the tree built with intronic sequences, this group is also
clearly apart from all of others (Electronic Supplementary
Material 4b) because the proportion of mutated sites
accumulated in the non-coding parts of these alleles is
equivalent to that found in the coding region (Electronic
Supplementary Materials 3 and 4).
Comparison of Mafa DRA cDNA alleles with EST libraries
We have produced DRA cDNA consensus sequences fromthree libraries obtained from one animal originating from
Malaysia (Osada et al. 2009; Table 4). In three ESTImmunogenetics (2010) 62:123–136

131

Fig. 3 Polymorphic sites of the full-length coding DRA cDNA in
macaques. Legend: The figure describes the Mafa-, Mamu-, Mane-,and Masy-DRA polymorphic sites (a) and corresponding amino acidchanges (b).The Mafa-DRA*01020101 sequence is used as reference.
Numbers attributed to each sequence correspond to the cDRA allele
numbering in Fig. 4 (phylogenetic network). The number 1 nucleotide
position corresponds to the first nucleotide of the start codon andamino acid numeration is based on IMGT numbering. Only
polymorphic sites are shown. Identity to the consensus is illustrated
by a dot. Non-synonymous polymorphic sites are underlined. LP,
leader peptide, α1 alpha 1 domain, α2 alpha 2 domain, C connecting

peptide, T trans-membrane domain. (1) Sequence not found in ourstudy, revealed in an Indonesian cynomolgus macaque [AM943637 by
Mee E.T. and Rose N.J. (unpublished)], (2) Full-length cDNA
deduced from Mamu-DRA genomic DNA sequence (AM910165,
Doxiadis et al. 2008). (3) Two expressed sequence tags (EST) of M.
nemestrina [Mane-DRA1 (EB520956) and Mane-DRA2 (EB521652)]
available in Genbank. (4) cDRA sequence of a homozygous M.
nemestrina (Mane-DRA01, GQ214407; this study).(5) Masy-DRA*01(EU877221) and Masy-DRA*02 (EU877222) sequences deduced fromtwo M. sylvanus animals (this study)

libraries, we obtained two consensuses strictly identicalwith Mafa-DRA*010212 and Mafa-DRA*02010101. The
two alleles are expressed at comparable levels. The DRB
cDNA sequences found in the three EST libraries differedfrom those of Mafa-DRB alleles (Mafa-DRB*W501, orMafa-DRB*W2101, or Mafa-DRB*W7201) belonging tohaplotypes #2a or #2b associated with Mafa-DRA*
02010101 in Mauritius and the Philippines, respectively.

polymorphic, especially in the amino acid positions which
are in contact with the peptide, while the DR alpha protein
is much less polymorphic. The number of DRB genes per
haploid genome is polymorphic in all mammal species
studied so far while the DRA gene is never duplicated. The
maintenance of the DRB gene polymorphism results from abalancing selection (Satta 1993). Although the DRA gene
polymorphism retained less attention than that of DRB, itsstudy is required to delineate the selective constraints which
influence the evolution of the DRA gene.
The aim of our study was to describe, in M. fascicularis
the polymorphism of the DRA gene. We studied 141 animals
representative of four populations either by characterizationof the DRA cDNA (78 animals) or partial DRA genomic
sequences (63 animals). In total, we characterized 22 MafaDRA cDNA alleles (18 of which had not been described

Discussion
The heterodimeric DR membrane proteins consist of acouple of DR alpha and DR beta proteins encoded by the
DRA and the DRB genes, respectively. In all mammal
species studied so far, the DR beta protein is highly

132

Immunogenetics (2010) 62:123–136

Human

6
16
686

28

357

26
1

339

748

697

7

46

19

657

2

591
46

10

315

253
253

549

253

13

22

11

558

657

253

12

27

591

198

667

21
17

3
315,
359

207
705
108,
315

20

23

21

15

14

41,63,87,
140,166,258

357

591

78

105,135

90

549

8

549,
591

253

90

46

174

9

18
105,135

590

90

24
591

5

705

25

4

29

30
Fig. 4 Phylogenetic network of the macaque cDNA sequences. The
network includes the Mafa-, Mamu-, Mane-, Masy-DRA nucleotide
sequences and was rooted by using the human HLA–DRA sequence as
outgroup. Each number noted in a circle represents the cDRA allele
and the other numbers correspond to the position of the polymorphic
sites on the full-length coding nucleotide sequence (position number 1
corresponds to the first nucleotide of the start codon). The correspon-

dence between numbers in circles and the DRA alleles is given in
Fig. 3. Two reticulations (polymorphic sites situated in positions 549
and 591), involving sequence numbers 28 (Mane-DRA2, EST) and 29(Mafa-DRA*02010101) are not shown in the phylogenetic network.
The Mafa-DRA*01020101 (noted 19 in the figure) probably corresponds to the macaque cDRA ancestral sequence

until now in M. fascicularis) and 41 partial (partial intron1
to partial intron3) Mafa-DRA genomic alleles. Mafa-DRA
polymorphism is higher than already reported in M. mulatta(the other macaque species which has been extensively
studied for cDRA polymorphism). Only 13 Mamu cDRA
alleles were described in the latter species (de Groot et al.
2004; Doxiadis et al. 2008), eight of which are identical to
22 Mafa-DRA described in this study.
Despite the large number of animals and diversity of
populations studied here, DRA gene polymorphism in M.
fascicularis is not completely defined by this study. Indeed,
the comparison of our cDRA sequence data set to the M.
fascicularis cDRA sequences available in databanks (including
the EST databanks), revealed one Indonesian cDRA allele(Mafa-DRA*01020301, Genbank access code AM943637)
which is missing in our full-length cDRA allele collection.
Moreover, the Mafa-DRA*010101 cDNA allele was foundonly in the Mauritius population, which was founded only 100
generations ago (Lawler et al. 1995). Hence, the probability

that Mafa-DRA*010101 derived from another allele bymutations is very faint. It is more likely that Mauritius
Mafa-DRA*010101 could have been brought by a founderanimal from a population incompletely studied here, such as
Java or Sumatra (Blancher et al. 2008; Tosi and Coke 2007).
Indeed, a systematic study of a larger range of population
samples including other M. fascicularis populations (various
continental populations and other island populations such as
from Sumatra, Borneo) would be required for an exhaustive
description of DRA polymorphism in this species.
In most pairwise comparisons between Mafa cDRA alleles,
mutations are synonymous with dN/dS ratios less than 1,
confirming that, in M. fascicularis, the DRA gene has been
submitted to a strong purifying selection (Hughes and Nei
1989). In the Mafa cDRA alleles neighbor-joining tree, theMafa-DRA*02010101 allele lies alone on a branch remotefrom all other Mafa cDRA alleles which are grouped with astar-like phylogeny. We estimated at about 12 million years
the time of divergence of the Mafa-DRA*02010101 allele

Immunogenetics (2010) 62:123–136

133

Fig. 5 Phylogenetic tree of macaque cDNA DRA sequences.
The tree of Mafa-, Mamu-,
Mane-, and Masy-cDRA alleles(734 bp) was obtained by meansof the neighbor-joining methodwith complete deletion option.
The tree was rooted with twooutgroups (HLA-DRA*0101 andPatr-DRA*10101). The percentage of replicate trees greaterthan 50% in which the associated taxa clustered together in thebootstrap test (1,000 replicates)
are shown next to the branches.
The evolutionary distances arein the units of the number ofbase substitutions per site. The
two Mane EST derived fromovary mRNA [Mane-DRA1(EB520956) and Mane-DRA2(EB521652)] were not includedin the neighbor-joining tree because they are not full-length.
However, they are clearlyhomologues of the MafaDRA*02010101 (see Fig. 3,
which describes the DRA cDNA
variable sites)

56

Mafa DRA*01020801 (EU877203)
Mamu DRA*010207 (AJ586880)
Mafa DRA*010218 (EU921294)
Mamu DRA*010401 (AJ586882)
63 Mafa DRA*01020901 (EU877205)
Mafa DRA*01020201 (EU877210)
Masy DRA*02 (EU877222)
Masy DRA*01 (EU877221)
Mafa DRA*010216 (EU921296)
Mafa DRA*010212 (EU877202)
51
Mamu DRA*010205 (AJ586878)
Mafa DRA*0109 (EU877213)
Mafa DRA*01020101 (EU877216)
Mamu DRA*010201 (AJ586874)
Mamu DRA*010202 (AJ586875)
Mamu DRA*0105 (AJ586884)
63 Mafa DRA*0106 (EU877208)
Mamu DRA*010302 (EU934772)
Mafa DRA*010304 (EU877207)
Mafa DRA*0105 (EU877214)
84 Mafa DRA*01040201 (EU877219)
Mamu DRA*010301 (AJ586881)
Mafa DRA*010301 (EU877212)
Mane DRA01 (GQ214407)
Mafa DRA*010305 (EU877206)
Mafa DRA*010302 (EU877211)
Mafa DRA*010303 (EU877209)
Mafa DRA*01020501 (EU877204)
Mafa DRA*01020701 (EU877217)
Mafa DRA*010101 (EU877215)
65 Mafa DRA*010105 (EU921295)
Mamu DRA*010204 (AJ586877)
Mamu DRA*010402 (AJ586883)
Mamu DRA*010203 (AJ586876)
Mafa DRA*01020301 (AM943637)
Mafa DRA*01010401 (EU877218)
Mamu DRA*010206 (AJ586879)
Mafa DRA*02010101 (EU877220)
100 Mamu DRA1*010601 (AM910165)
HLA DRA*0101 (AL935032)
Patr DRA1*0101 (AM910166)
100

0.005
from the most recent common ancestor of all Mafa cDRA
alleles. This divergence time largely precedes macaquespecies radiation in Asia which could explain that homologues of Mafa-DRA*02010101 are found in two othermacaque species: M. mulatta (Mamu-DRA1*010601) andMacaca nemestrina (Fig. 3). This allele sharing is mostprobably the result of trans-species evolution (Klein 1987),
although introgression could be considered as an alternative
hypothesis (Bonhomme et al. 2009; Street et al. 2007).
It remains to be explained how this allele could persist forso long in three different species. One of the most probablecauses of this persistence is balancing selection which might
result from functional difference between the allele MafaDRA*02010101 and the other Mafa-DRA alleles. MafaTable 4 Number of EST
corresponding to the two DRA
alleles of a Malaysian cynomolgus monkey in EST libraries
derived from three lymphoidorgansDRA*02010101 differs from all other Mafa-DRA alleles by
eight specific mutations among which four are nonsynonymous. One amino acid change, present in the leader
peptide is not conservative (Ile->Thr) and could have an
impact on the exportation of the DR alpha protein
influencing the density of peptide-presenting DRA–DRB
heterodimers at the surface of the presenting cells. The threeother amino acid changes, Glu-4-Asp, Phe-22-Tyr, and Ile31-Leu, are conservative, located in the alpha-1 domain, andonly the two latter involve amino acid residues which are incontact with the peptide presented by the DRA–DRB
heterodimer (Reche and Reinherz 2003). Although thesesubstitutions are conservative, they could have an impact on
the functional properties of the DR peptide groove.
Mafa-DRAEST libraries
SpleenThymusBone marrow

QspA,Lib.23009, n=9,783
QthA,Lib.24021, n=9,566
QbmA,Lib.22708, n=9,472
Total EST

*010212*02010101

40
26
17
83

32
25
14
71

134

On the other hand, the mutations described here in thecoding region of Mafa-DRA*02010101 (as well as intronicmutations) could have accumulated through a hitchhiking
effect related to distant mutations in the DRA genepromoter region or in the DRB gene, for example. Indeed,mutation in the promoter region could affect the transcription level of the gene. To explore this eventuality, we
studied the representation of two DRA alleles in three EST
libraries deriving from mRNA of three lymphoid organs of
one Malaysian DRA-heterozygous animal (MafaDRA*02010101/Mafa-DRA*010212). The numbers of
EST on the two alleles do not differ in the three organs
analyzed (Table 4), and we concluded that the level oftranscription of the allele Mafa-DRA*02010101 does notdiffer significantly from that of Mafa-DRA*010212.
In total, in M. fascicularis, from the results of the present
study and those available in the literature, 30 cDRA alleles arecharacterized and defined by 29 polymorphic positions. This
polymorphism is much higher than in humans. For example,
in the Philippine population, we found 11 Mafa-DRA alleles
and 13 Mafa-DRB haplotypes. In humans, only five HLA–DRA alleles are known [two, named HLA-DRA*0101 andHLA-DRA*010202, and three revealed by the study of 58
unrelated individuals (Voorter and Van den Berg-Loonen2006)] while more than 700 HLA–DRB1 alleles (defined only
on exon 2) have been described. Although, the HLA–DRA
gene has not been studied in some large samples in diverse
populations, it is unlikely that HLA–DRA gene polymorphism
reaches a level comparable to that of the Mafa-DRA gene.
Although the polymorphism of the Mafa-DRA gene ishigher than that observed in humans, it is clearly lower thanthat observed in Mafa-DRB genes. Further studies areneeded to clarify the functional constraints that result in the
predominance of purifying selection on the DRA gene while
the DRB genes are under diversifying selection.
Acknowledgments This work was performed with funds from the
French Ministry of Research (EA3034 contract), from a research grantfrom Novartis, and with the help of ANRS (contract number 07375).
Other funds were from the Laboratory of Dr. Takashi Shiina. We thankPr. Brigitte Le Mauf (Nantes, France), Dominic Borie (Stanford,
USA), Eric André (Bioprim, Baziège, France), Marc Bigaud(Novartis, Bâle), Dr. Jacques Ducos de Lahitte (Ecole Nationale
Vétérinaire de Toulouse), for their help in obtaining macaque blood
samples. Our thanks are also due to Peter Winterton for his
contribution in drawing up the final version of this manuscript.
We are pleased to thank all the technicians of the Toulouse Laboratoryof Immunogenetics: Béatrice Atlan, Audrey Dauba, Stéphanie DespiauSchiavinato, and Sylvie Hébrard for their excellent technical assistance.

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