J. Renquin
A. Sanchez-Mazas
L. Halle
S. Rivalland
G. Jaeger
K. Mbayo
F. Bianchi
C. Kaplan
Key words:
Aka Pygmies; Africa; anthropology; Bantu
Congolese; genetic diversity; HLA class II;
linguistics; MHC selection; PCR-dot blot reverse;
PCR-SSP
Acknowledgments:
This work was supported by a Swiss FNRS grant
(.3100-49771.96) to A. Sanchez-Mazas and by
the French DGRST (No. 77.7206; 78.7.2456;
79.7.0456).
HLA class II polymorphism in Aka Pygmies
and Bantu Congolese and a reassessment of
HLA-DRB1 African diversity
Abstract: HLA-DRB1, -DQB1 and -DPB1 polymorphisms were investigated in two African populations, the Basse Lobaye Aka Pygmies of the
Central African Republic, and a Bantu-speaking group from the Democratic
Republic of Congo Kinshasa. Allelic and haplotypic frequency distributions
reveal marked differences between the two populations in spite of their
geographical proximity: the Aka exhibit high frequencies for several alleles,
especially at the DPB1 locus (0.695 for DPB1*0402), probably due to rapid
genetic drift, while the Bantu distributions are more even. Genetic distances
computed from DRB1 allelic frequencies among 21 populations from North
and sub-Saharan Africa were applied to a multidimensional scaling analysis.
African populations genetic structure is significantly shaped by linguistic
differentiation, as confirmed by an analysis of molecular variance. However,
selective neutrality tests indicate that many African populations exhibit an
excess of heterozygotes for DRB1, which is likely to explain the genetic similarity observed between some North African and Bantu populations. Overall, this study shows that natural selection must be taken into account when
interpreting the patterns of HLA diversity, but that this effect is probably
minor in relation to the stochastic events of human population differentiations.
Authors’ affiliations:
J. Renquin1,
A. Sanchez-Mazas1,
L. Halle2,
S. Rivalland2,
G. Jaeger3,
K. Mbayo4,
F. Bianchi2,
C. Kaplan2
1
Laboratory of Genetics and
Biometry (LGB), Department
of Anthropology and Ecology,
University of Geneva,
Geneva, Switzerland,
2
Institut National de la
Transfusion Sanguine
(INTS), Laboratoire
d’Immunologie Plaquettaire,
Paris, France,
3
Centres Européens
Associées de Biologie
Humaine (CEABH), Paris,
France,
4
Cliniques Universitaires de
Kinshasa, Kinshasa,
Democratic Republic of
Congo
Due to its high level of polymorphism revealed by high-resolution
DNA molecular typings, the human major histocompatibility complex (MHC) plays a key role in anthropology for investigating the
genetic relationships between populations. Previous studies related
to HLA diversity in sub-Saharan Africa indicate a high level of
heterogeneity among populations within this continent, and a correlation with geographic and/or linguistic differentiations (1–5). However, some populations, like the Pygmies, were not represented. The
Received 13 July
accepted for publication 17 September 2001
Copyright c Munksgaard 2001
Tissue Antigens . ISSN 0001-2815
Tissue Antigens 2001: 58: 211–222
Printed in Denmark . All rights reserved
aim of this study is to investigate the HLA class II polymorphism in
two African populations: Aka Pygmies (6) from the Central African
Correspondence to:
Department of Anthropology
and Ecology
12 rue Gustave-Revilliod
1227 Geneva
Switzerland
Tel: π41 22 702 69 84
Fax: π41 22 300 03 51
e-mail: alicia.sanchezmazas/anthro.unige.ch
or
Cécile Kaplan
INTS
6 rue Alexandre Cabanel
75739 Paris
France
Tel: π331 4449 3067
Fax: π331 4567 1930
e-mail:
cecile.kaplan/teaser.fr
Republic (CAR), and a group of Bantu individuals originating from
the Democratic Republic of Congo Kinshasa (DRC).
211
Renquin et al : HLA class II in Aka Pygmies and Congolese
Fig. 1. Present geographic location of the Aka Pygmies and the Bantu Congolese under study.
(Kinshasa) to Congo (Brazzaville) and the second, approximately 150
Aka Pygmies
years ago, left Congo (Brazzaville) to reach the village of Bagandu
From 1975 to 1980, the EurAfrican Centre for Human Biology (CE-
situated in the southwest of the CAR.
ABH) in cooperation with the Central African government, under-
An initial study, published by the INTS group in 1981 (9) on
took a comprehensive study of the Aka Pygmies (Basse Lobaye
HLA class I serological typing revealed the presence of HLA-B37
region) and their environment (7). This population is of particular
(8%) with A30-B37 linkage disequilibrium (D⫽0.035, PⰆ0.001).
interest because it constitutes an isolate of nomadic hunter-gath-
This allele is very rare or absent in other sub-Saharan populations
erers in a primary environment. These Pygmies move around con-
except the Fulani (17%) (4). Sixty samples typed again in 1996
stantly in the southern part of Central Africa and the northern part
revealed the presence of B41 (2.44%) and B42 (3.44%).
of the People’s Republic of Congo Brazzaville (8). Their territory (3æ5
N and 17æ30–18æ E) is defined by the natural borders of the Oubang-
Bantu Congolese
ui river and its two tributaries the Lobaye and the Ibanga. Every
year the Aka leave their environment for a few weeks to work for
The Democratic Republic of Congo Kinshasa (DRC) covers an
the Bantu population in exchange for fulfilment of their needs. Ac-
area of some 2,400,000 square kilometres stretched out between a
cording to the Pygmies, they have been two waves of migration:
latitude of 5æ2ø north and a latitude of 13æ15ø south. Because of
the original older migrant group travelled eastward from Congo
their geographical situation in the centre of Africa, the autoch-
212
Tissue Antigens 2001: 58: 211–222
Renquin et al : HLA class II in Aka Pygmies and Congolese
thonous population and more specifically the Pygmies were faced
(14) was used for samples with at least 106 of PBMC, and the Pro-
with Bantu migration waves towards the end of the first milleni-
teinase K lysis technique (15) was used for samples with less than
um (10). These Bantus migrated along 3 paths: the eastern route,
106 of PBMC.
leading to settling in savannahs of the Katanga and Kivu re-
HLA class II alleles for loci DRB1, DQB1 and DPB1 were deter-
gions, the western route, resulting in settlement in the Lower
mined by polymerase chain reaction (PCR)-reverse dot blot hybridi-
Congo and Bandundu regions, and finally the central route, end-
zation INNO-LIPA (Innogenetics N.V., Zwijnaarde, Belgium) (16).
ing in the Equator and Bandundu regions. Later came Sudanese
DQA1 and DRB1*15 high-resolution were analysed by PCR-SSP (17)
migration to the north-eastern part of the country (Ubangi-Kibali-
(Dynal France SA., Compiègne France). The HLA nomenclature was
Ituri) leading to interbreeding with the Bantus. Finally, coming
used according to S.G.E. Marsh for the WHO Nomenclature Com-
from the Ethiopian plateaux, Nilotics travelled up the Nile valley
mittee for Factors of the HLA system (January 2001 update; http://
to settle near the great lakes in the Kivu region. The Congolese
www.anthonynolan.com/HIG/nomenc.htm/).
included in this study (Fig. 1) belong to the Bantu-speaking
group (11, 12) which is 35% composed of Kongo, 29% of Luba
(75% from the provinces of Eastern-Kasaı̈ and 25% from Katan-
Statistical analyses
ga), 16% of Mongo, 8% of Yaka, 6% of Yanzi and 4% of Northwest Bantu (Sakata Teke). This work follows an initial study of
Allele and haplotype frequencies were estimated by an EM algo-
the markers HLA class I and class II typed by serological tech-
rithm (18) using the computer package Arlequin (19). The hypoth-
niques in 1994 (13).
esis of Hardy-Weinberg equilibrium was tested by two independent
The geographic location of the two sampled populations is
shown in Fig. 1.
approaches (exact test and Chi-square) as explained by SanchezMazas et al. (20). Two-locus haplotypes were tested for linkage disequilibrium. For each haplotype AB, we computed the normalised
linkage disequilibrium D’AB, and we tested the null hypothesis of
Material and methods
no linkage disequilibrium by c2 ⫽
2nD2AB
(1 degree of
pA(1ªpA)pB(1ªpB)
freedom), where 2n is the number of gametes (21). Global linkage
Population samples
disequilibrium between each pair of loci was tested both by a Chisquare and a likelihood ratio test (both tests implemented in Arle-
Pygmies
quin). Allelic and haplotypic frequencies observed in the sampled
Blood samples were taken from 543 Pygmies and were transported
populations were compared to those estimated in the North and
to Paris within 36 h. The lymphocytes were separated at National
sub-Saharan African population samples available at the 12th Inter-
Institute of Blood Transfusion (INTS) using ficoll hypaque density
national Histocompatibility Workshop and later publications (4, 5,
gradient centrifugation and then cryopreserved in liquid nitrogen.
22–29) (Sanchez-Mazas, personal results). For that purpose, pair-
A total of 93 samples were taken from unrelated Aka Pygmies,
wise linearized Fst’s or co-ancestry coefficients (30) were computed
including both sexes, and covering the 20 to 70 age group. The
a) on the basis of a limited number of DRB1 alleles (13 broad speci-
samples were chosen without common ancestry going back at least
ficities plus a null allele) to allow the comparison of many popula-
three generations.
tions (n⫽21 African samples), and b) on the basis of most DRB1
alleles (65 DRB1 subtypes) for a reduced set of 13 populations. The
choice of populations was done on the basis of high sample size
Congolese
and, for the second analysis, high resolution of DRB1 typings. Gen-
Samples from 85 unrelated healthy volunteer blood donors living in
etic distances (co-ancestry coefficients) were applied to a multidi-
Kinshasa or its immediate surroundings were collected on EDTA.
mensional scaling analysis by using the NTSYS computer package
These were shipped to Paris for DNA extraction.
(31). Fst values were tested for significance by a permutation procedure, and were used to investigate population genetic structure
DNA extraction and HLA molecular typing
by an analysis of variance (ANOVA) (32). Selective neutrality of
DRB1 distributions was tested in all compared populations by
Genomic DNA was extracted from frozen peripheral blood cells
Ewens-Watterson’s (33, 34) and Slatkin’s (35, 36) statistical tests. All
(PBMC) by two different techniques: the Miller modified method
these tests were carried out by using Arlequin.
Tissue Antigens 2001: 58: 211–222
213
Renquin et al : HLA class II in Aka Pygmies and Congolese
Allelic frequencies and Hardy-Weinberg equilibrium significance in Aka Pygmies and Bantu Congolese
Aka Pygmies
(n⫽93)
Bantu Congolese
(n⫽85)
DQB1*0501
0.218
0.171
DRB1*0101
0.005
0.018
DQB1*0502
0.000
0.012
DRB1*0102
0.000
0.041
DQB1*05031
0.000
0.012
DRB1*0301
0.118
0.065
DQB1*0601
0.013
0.000
DRB1*0302
0.000
0.029
DQB1*0602
0.182
0.299
DRB1*0401
0.000
0.012
DQB1*0603
0.038
0.006
DRB1*0403
0.005
0.000
DQB1*0604
0.064
0.035
DRB1*0405
0.011
0.012
DQB1*0605
0.000
0.012
DRB1*0406
0.000
0.006
DQB1*0606
0.005
0.000
DRB1*0701
0.241
0.065
DQB1*0608
0.011
0.000
DRB1*08
0.000
0.053
DQB1*0609
0.000
0.032
DRB1*0901
0.011
0.053
null
0.009
0.023
DRB1*1001
0.011
0.018
DRB1*11
0.065
(0.1821)
A) DRB1
DRB1*1101
0.094
DRB1*1102
0.059
DRB1*1104
0.029
DRB1*1201
0.129
0.029
DRB1*1202
0.016
0.000
DRB1*1301
0.161
0.065
DRB1*1302
0.048
0.111
DRB1*1303
0.000
0.006
DRB1*1308
0.000
0.006
DRB1*1401
0.038
0.006
DRB1*15
(0.1301)
0.205
DRB1*1502
0.007
DRB1*1503
0.123
DRB1*16
DRB1*1601
null
0.018
0.005
0.006
0.000
HWE2: P-value by exact test
0.15 (n.s.)
0.42 (n.s.)
HWE: P-value by c2 test
5.10ª10(3)
0.94 (n.s.)
DQB1*02
0.353
0.171
DQB1*0301
0.043
0.168
DQB1*0302
0.032
0.006
DQB1*03032
0.021
0.018
DQB1*0402
0.011
0.035
B) DQB1
1
2
3
4
Aka Pygmies
(n⫽93)
HWE: P-value by exact test
0.16 (n.s.)
0.13 (n.s.)
HWE: P-value by c2 test
0.14 (n.s.)
0.78 (n.s.)
C) DPB14
(n⫽81)
(n⫽83)
DPB1*0101
0.049
0.229
DPB1*0201
0.030
0.175
DPB1*0301
0.025
0.060
DPB1*0401
0.031
0.054
DPB1*0402
0.695
0.199
DPB1*1101
0.000
0.030
DPB1*1301
0.006
0.048
DPB1*1401
0.000
0.006
DPB1*1701
0.000
0.042
DPB1*1801
0.049
0.073
DPB1*1901
0.006
0.012
DPB1*2901
0.000
0.006
DPB1*3201
0.000
0.006
DPB1*3401
0.006
0.000
DPB1*3901
0.068
0.012
DPB1*4001
0.031
0.012
DPB1*4901
0.000
0.024
DPB1*5101
0.000
0.006
DPB1*5501
0.000
0.006
null
0.004
0.000
0.73 (n.s.)
0.39 (n.s.)
0.99 (n.s.)
0.99 (n.s.)
HWE: P-value by exact test
214
Tissue Antigens 2001: 58: 211–222
2
HWE: P-value by c test
Computed as the sum of the subtypes frequencies
HWE: Hardy-Weinberg equilibrium hypothesis
P∞0.001 but two rare phenotypes (DRB1 1001/0101 and DRB1 1502) account for 68% of the total c2
Not the same sample sizes for DPB1 as for DRB1 and DQB1
Table 1
Bantu Congolese
(n⫽85)
Renquin et al : HLA class II in Aka Pygmies and Congolese
tively). Finally, a drastic difference is observed for allele DPB1*0402,
Results and discussion
as the frequency found in the Aka reaches 69.5% (against 19.9%
in the Congolese). More even frequencies characterise the DPB1 dis-
Allelic frequencies and Hardy-Weinberg equilibrium
tribution estimated in the Congolese. Overall, the Aka exhibit a
Allele frequencies are presented in Table 1. The hypothesis of
lower level of HLA genetic diversity than the Bantu, and especially
Hardy-Weinberg equilibrium can be accepted at all loci (DRB1,
so at the DPB1 locus (observed heterozygosities in Aka versus Con-
DQB1, DPB1) in both Aka and Bantu Congolese. This result is quite
golese: 86.1% vs. 90.9% at the DRB1 locus, 78.6% vs. 81.9% at the
surprising for the Congolese sample, which is constituted of individ-
DQB1 locus, and 50.4% vs. 85.9% at the DPB1 locus).
uals coming from many different tribes. It suggests that the test for
For some alleles, the frequencies observed in the Aka are in fact
Hardy-Weinberg equilibrium is conservative, but also that Bantu
close to those found in East Africans: DRB1*0701 and DQB1*02
populations are genetically homogeneous.
are frequent in Oromo and Amhara (where the frequency of
In spite of being geographically close, the two populations ex-
DRB1*0701 is about 20%, and that of DQB1*02 more than 30%).
hibit marked differences in their allelic profile. The most contrasted
For other alleles, Aka frequencies are closer to those found in Bantu
frequencies are those found for DRB1*0701, 0301, 1201 and 1301,
populations: DRB1*1301 (16% in the Aka) is frequent in the Bubi
much higher in the Aka (24.1%, 11.8%, 12.9% and 16.1%, respec-
(23%), whereas another DRB13 subtype, DRB1*1302, is frequent in
tively), as well as DRB1*15, 11 and 1302, predominant in the Congo-
East Africans, and DQB1*0501 (22% in the Aka) is generally fre-
lese (20.6%, 18.2%, and 11.2%, respectively). At the DQB1 locus,
quent in the Bantu (15–20%). From these results, it is not clear
we mostly note higher DQB1*02 and 0501 and lower (although high)
whether the Aka are genetically closer to Afroasiatic or Bantu popu-
DQB1*0602 frequencies in the Aka (35.3%, 21.8%, 18.2%, respec-
lations.
DRB1-DQB1 most frequent haplotypes (frequency ⬎3% in at least one population) and linkage disequilibrium
in Aka Pygmies and Bantu Congolese
Aka Pygmies (n⫽93)
Dø
Bantu Congolese (n⫽85)
2
c
DRB1-DQB1
Frequency
0102–0501
–
–
0301–02
0.112
0.929
0701–02
0.219
0.863
08–0301
–
–
0901–02
0.011
1.000
11–0301
0.043
1.000
1101–0301
–
–
1102–0301
–
–
1104–0301
–
11–0602
0.011
1101–0602
–
–
1201–0501
0.129
1.000
1301–0602
0.059
0.226
1301–0603
0.032
0.830
1301–0604
0.032
0.404
4.27
1302–0501
0.011
0.005
⬍0.001
1302–0604
0.032
0.644
25.3
1401–0501
0.032
0.817
15–0602
–
–
1503–0602
0.112
0.893
–
ª0.084
Table 2
Frequency
–
0.035
0.827
9.59
**
11.2
**
0.065
1.000
22.02
**
19.8
**
0.057
0.849
15.87
**
–
–
0.047
0.866
13.92
**
1.19
n.s.
0.053
1.000
18.01
**
**
–
–
–
–
0.024
0.099
–
–
0.059
1
–
54.3
Dø
c2
Significance
Significance
–
–
0.32
n.s.
20.59
**
–
–
0.012
0.279
0.8
*
0.008
n.s.
–
–
–
–
–
–
0.071
0.643
5.45
*
**
0.021
0.638
4.07
*
n.s.
0.018
ª0.087
0.01
n.s.
**
0.006
1.000
6.76
**
*
–
–
–
n.s.
0.062
0.460
8.05
**
**
0.018
0.437
4.05
*
6.56
*
–
–
–
–
–
–
0.200
0.959
**
–
–
33.5
2.82
10.5
33.6
–
26.5
–
**
–
**P⬍0.01; *P⬍0.05; frequencies in bold: ⬎10%.
Tissue Antigens 2001: 58: 211–222
215
Renquin et al : HLA class II in Aka Pygmies and Congolese
DRB1-DQB1-DPB1 most frequent haplotypes (frequency ⬎3%) and linkage disequilibrium significance in Aka Pygmies and Bantu Congolese
Genetic distances among African populations
Linkage disequibrium significance
The multidimensional scaling analyses (MDS) based on DRB1 gen-
Frequency
DRB1-DQB1
DQB1-DPB1
DRB1-DPB1
etic distances and carried out on the broad (21 populations) and
0701–02–0402
0.216
**
n.s.
n.s.
shown in Figs. 2 and 3, respectively. An MDS analysis carried out
1201–0501–0402
0.111
**
n.s.
n.s.
on DRB1-DQB1 haplotypes on 18 populations gives very close re-
03011–02–0402
0.075
**
n.s.
n.s.
sults to those presented in Fig. 2 (results not shown).
1503–0602–0402
0.072
**
n.s.
n.s.
Except for the Aka, the populations were clustered according to
11–0301–0402
0.037
**
n.s.
n.s.
the linguistic phylum to which their language belongs. The Merina
1301–0602–3901
0.031
n.s.
**
n.s.
from Madagascar are peculiar as they speak an Austronesian lan-
1503–0602–3901
0.031
**
**
*
guage of Asian origin. They are clearly discriminated genetically
DRB1-DQB1-DPB1
haplotype
reduced (13 populations) data sets (see Material and methods) are
A) Aka Pygmies (n⫽81)
from all other African populations due to the main contribution of
B) Bantu Congolese (n⫽83)
15–0602–0201
0.100
**
*
**
South-East Asian ancestors to their present genetic pool (39). In
15–0602–0402
0.056
**
n.s.
n.s.
spite of their relative overlap, speakers of the other linguistic groups
1101–0602–0101
0.030
*
n.s.
n.s.
represented (Afroasiatic, Niger-Congo, Khoisan) can be discrimi-
**P⬍0.01; *P⬍0.05; n.s. not significant
nated genetically from each other, suggesting a significant corre-
Table 3
lation between genetic and linguistic differentiations, in keeping
with previous results (1, 4, 25). This is confirmed statistically here
by the ANOVA analysis, which shows that the variance due to differentiations among linguistic groups, Fct, is highly significant
Haplotype frequencies and linkage disequilibrium
(Fct⫽0.016, PⰆ0.0001) (the Aka were included in the Niger-Congo
cluster for this analysis). A reasonable interpretation of the corre-
In Tables 2 and 3 are presented the estimated frequencies and link-
lation found between genetic and linguistic diversity in Africa can
age disequilibrium significance for DRB1-DQB1 and DRB1-DQB1-
be found in previous publications (2, 3, 5).
DPB1 haplotypes, respectively. The frequency differences observed
in Table 2 between Aka and Bantu Congolese are in keeping with
However, we also note variable degrees of genetic heterogeneity
within each linguistic group:
what we expected from the observed allelic frequencies (Table 1)
and the tight linkage disequilibrium generally found between loci
DRB1 and DQB1 (37, 38).
– among the Afroasiatic, the Ethiopian Oromo and Amhara (subSaharan populations) are grouped together and genetically differ-
According to Table 3A, the 4 most frequent DRB1-DQB1 haplo-
entiated from the North African populations. Geographic differen-
types found in the Aka are observed in combination with
tiation, with limited gene flow, on both sides of the Sahara Desert
DPB1*0402. This is clearly due to the extremely high frequency
may explain this result.
observed for this allele (almost 70%), rather than to linkage disequi-
– among the Khoisan, the San, with a very high frequency of
librium. Indeed, linkage disequilibrium is found to be non signifi-
DRB1*0401 (42%), are highly differentiated from the Khoi and
cant between DPB1 and the other loci for most haplotypes (Table
from all other populations, probably as a result of a rapid genetic
3). Moreover, global tests of linkage disequilibrium indicate a sig-
drift. On the other hand, the Khoi exhibit some genetic affinities
nificant association between DRB1 and DQB1 in both populations
with Afroasiatics, and more particularly with the East Africans
(PⰆ0.001 by both methods in both populations), while only one of
Oromo and Amhara (high frequency of DRB1*1302 and DRB*15),
the 8 tests of linkage disequilibrium involving DPB1 (2 methods,
as shown in Figs. 2 and 3. This corroborates the hypothesis that
applied to the 2 loci pairs DRB1-DPB1 and DQB1-DPB1, in the 2
the ancient Khoisan homeland was much more extended than
populations) is found significant (DQB1-DPB1 by the exact test in
today and included East Africa (2). Contrary to the San, the Khoi
the Aka). The almost independence of DPB1 relatively to the other
adopted pastoralism and probably kept more contacts with their
HLA loci has been suggested before (38).
neighbors.
As noted above for DPB1 taken alone, the DRB1-DQB1-DPB1
– among the Niger-Congo, the West African Mandenka and the
distribution observed in the Congolese is more even than in the Aka
Bubi (who live on an island at an intermediate location between
(Table 3B).
western and southern Africa), are both genetically close to each
216
Tissue Antigens 2001: 58: 211–222
Renquin et al : HLA class II in Aka Pygmies and Congolese
Fig. 2. Multidimensional scaling analysis of 21 African populations based on the frequencies of 14 DRB1 allele frequencies
(DRB1*01, 03, 04, 07, 08, 09, 10, 11, 12, 13, 14, 15, 16, null).
Population samples are: 93 Aka Pygmies from the Central African Republic
(present study), 85 Bantu Congolese for Congo Kinshasa (present study),
198 Mandenka from Eastern Senegal (5), 101 Bubi from the island of Bioko,
Equatorial Guinea (22)*, 167 Banzabi from Gabon (24)*, 72 Matabele and
82 Shona from Zimbabwe, 199 Zulu from South Africa, 75 Sotho from Lesotho, 163 Merina from Madagascar (4)*, 107 M’Zab from Algeria, 101 Beduin
from Siwa (Egypt), 119 Egyptians (Mansoura), 107 Egyptians (Assouan), 99
Tunisian (25)*, 101 Algerian (25, 26), 98 Moroccan (25*, 27), 98 Amhara
and 83 Oromo from Ethiopia (28)*, 77 San and 91 Khoi from Namibia (29).
Population names are bolded and/or underlined according to the results of
Ewens-Watterson’s and Slatkin’s tests for selective neutrality. Bold: P⬍0.05
by both tests; underlined: P⬍0.05 by one test and P⬎0.05 by the other test;
neither bolded nor underlined: P⬎0.05 by both tests. Non-significant Fst’s
are found for the following population pairs: a) P⬎0.05: Amhara-Oromo,
Algerian-Moroccan, Algerian-Tunisian, Moroccan-Tunisian, Sotho-Matabele, Matabele-M’Zab, Egyptian (South)-Beduin; b) 0.01⬍P⬍0.05: Sotho-Zulu,
Sotho-Shona, Sotho-Algerian, Sotho, Egyptian (Delta), Sotho-Egyptian
(South), Sotho-Beduin, Congolese-Matabele, Congolese-Beduin, CongoleseBanzabi, Zulu-Matabele, Zulu-Tunisian, Matabele-Beduin, Algerian-Egyptian (South), Algerian-Beduin.
*Raw data from XIIth International Histocompatibility Workshop and Conference, personal communication to A. Sanchez-Mazas.
other and differentiated from the other populations (Fig. 2). This
ment with geography. Coming back to Fig. 2, we note that most
is due to a high frequency of DRB*13 (37%). However, the
Bantu populations (Matabele, Shona, Sotho, Zulu, Banzabi and
DRB*13 subtype involved is in fact not the same in the two popu-
Congolese) are genetically close to each other, in keeping with
lations: DRB1*1304 is the most frequent in the Mandenka (25%
their common origin. On the other hand, they are also close to
(5)), and DRB1*1301 the most frequent in the Bubi (23% (22)).
Afroasiatics, and, more particularly, North Africans. As indicated
This is revealed by Fig. 3 (where fewer populations but more
in the Legends for Figs. 2 and 3, significance tests on Fst’s be-
HLA alleles are considered), showing that the Bubi are genetically
tween populations indeed reveal many non-significant values
intermediate between the Mandenka and the Congolese, in agree-
among Bantu and North Africans. This unexpected result is hardTissue Antigens 2001: 58: 211–222
217
Renquin et al : HLA class II in Aka Pygmies and Congolese
Fig. 3. Multidimensional scaling analysis of 13 African populations based on the frequencies of 65 DRB1 allele frequencies
(DRB1*0101, 0102, 0103, 0104, 0301, 0302, 0303, 0304, 0305,
0401, 0402, 0403, 0404, 0405, 0406, 0407, 0408, 0410, 0412,
0413, 0414, 0417, 0422, 07, 08, 0901, 1001, 1101, 1102, 1103,
1104, 1105, 1107, 1109, 1113, 1116, 1117, 1201, 1202, 1301,
1302, 1303, 1304, 1305, 1307, 1308, 1309, 1310, 1311, 1312,
1314, 1315, 1319, 1401, 1402, 1403, 1404, 1407, 1408, 1409,
1414, 1418, 15, 16, null). Population samples are listed in the Legend
for Fig. 2. Population names are underlined according to the results of
Ewens-Watterson’s and Slatkin’s tests for selective neutrality. Underlined:
P⬍0.05 by one test and P⬎0.05 by the other test; not underlined: P⬎0.05
by both tests.
ly explained by historical relationships, and may involve selective
populations. Pygmies are presently scattered across many regions
effects, as discussed below. Finally, the Aka Pygmies, although
of Central Africa, with some groups (either Pygmies or ‘‘Pygmo-
included in the Bantu cluster from a linguistic point-of-view, are
ids’’) living in eastern regions located in Sudan, Uganda and Ethi-
genetically isolated. This is due, for example, to their high fre-
opia (6). They probably inhabited a larger part of Central Africa
quency of DRB1*1201, very rare in other populations. Their gen-
before, and underwent several migrations between eastern and
etic divergence is easily explained by their peculiar mode of life
western regions.
(as hunters-gatherers) and their low population size (small tribes),
which probably lead to a rapid genetic drift. However, they also
Neutrality tests
share genetic affinities with both Afroasiatics and Bantu, as mentioned before on the basis of allelic frequencies. Besides a possible
Both tests of selective neutrality (Ewens-Watterson’s and Slatkin’s)
convergence effect due to natural selection, as explained below, it
used in the present study give non-significant results at the 3 HLA
is thus likely that the Aka Pygmies, who now speak a Bantu
class II loci for both Aka and Congolese (all P⬎0.05). This is not
language, were subject to some admixture with neighboring
remarkable for DPB1, which generally exhibits skewed and neutral-
218
Tissue Antigens 2001: 58: 211–222
Renquin et al : HLA class II in Aka Pygmies and Congolese
like frequency distributions (37, 38, 40). At the other HLA class II
populations projected at the periphery of the multidimensional scal-
loci, the present results confirm that rejection of the neutral hypoth-
ing analysis exhibit a neutral-like DRB1 distribution, while the
esis is not systematic.
populations projected at the centre, where Afroasiatic and Bantu
We also applied neutrality tests to the DRB1 distributions of all
linguistic groups overlap, exhibit an excess of heterozygotes. From
African populations included in our genetic distance analyses. The
these results, we may suggest that balancing selection, already
results are given in Table 4 and reported in Figs. 2 and 3, where
shown to be acting on MHC (5, 20, 37, 38, 41), maintained a high
the populations for which we observe a significant departure from
level of polymorphism in many populations, often leading to ap-
neutrality due to a lower homozygosity are bolded and/or under-
parent genetic affinities between populations which are distantly
lined.
related from a historical and/or a geographical point-of-view. This
According to Fig. 2, where we consider many populations but a
would explain in part the genetic proximity of Afroasiatics and
limited number of alleles, most Bantu and Afroasiatic populations
Bantu. On the other hand, the results are quite different when we
exhibit a significant excess of heterozygotes. The exceptions are the
consider a lower number of populations and a maximal number of
Banzabi (Bantu from Gabon (24)), the Mandenka (5), both Khoisan
alleles (Table 4 and Fig. 3): only non-significant or contradictory
populations, and the Aka Pygmies. For some populations (Bubi,
results are observed. We note from Table 4 that the fact of including
Matabele, Beduin, Oromo, Tunisian, and Merina), Ewens-Watter-
a large number of alleles reduces the expected homozygosity, while
son’s and Slatkin’s tests give contradictory results (one rejection and
the observed homozygosity does not change substantially. The rejec-
one acceptance of the null hypothesis). Overall, we note that the
tion of the neutral hypothesis, in the first analysis, may be biased
Results of Ewens-Watterson’s and Slatkin’s selective neutrality tests applied to the DRB1 frequency distributions of 21 African population samples
DRB1 broad specificities (max. number⫽14)
Populations
N
n
Fobs
DRB1 subtypes (max. number⫽65)
Fexp
PEW
PSL
N
n
Fobs
Fexp
PEW
PSL
0.543
Aka Pygmies
93
12
0.162
0.255
0.082
0.255
93
17
0.139
0.183
0.236
Bantu Congolese
85
13
0.136
0.238
0.047
0.028
85
22
0.091
0.134
0.094
0.050
Mandenka
196
13
0.207
0.280
0.263
0.118
196
22
0.115
0.166
0.141
0.109
Bubi
101
13
0.194
0.251
0.312
0.042
101
20
0.097
0.157
0.051
0.006
San
77
11
0.277
0.267
0.648
0.600
77
14
0.221
0.210
0.666
0.417
Khoi
91
13
0.176
0.241
0.238
0.165
91
19
0.139
0.163
0.395
0.155
Amhara
98
10
0.145
0.319
0.005
0.005
98
20
0.117
0.154
0.210
0.336
Oromo
Tunisians
83
11
0.156
0.279
0.034
0.092
83
19
0.116
0.157
0.207
0.133
101
13
0.135
0.243
0.026
0.056
101
26
0.092
0.114
0.262
0.040
Algerians
100
13
0.121
0.248
0.009
0.008
100
28
0.077
0.102
0.150
0.054
Egyptians (Delta)
119
14
0.126
0.239
0.035
0.017
119
52
0.055
0.050
0.776
0.732
Egyptians (South)
107
14
0.124
0.230
0.021
0.014
107
37
0.060
0.075
0.231
0.223
Beduin
102
13
0.135
0.245
0.023
0.054
102
27
0.083
0.112
0.163
0.140
Sotho
75
13
0.112
0.227
0.003
0.017
Shona
82
14
0.127
0.221
0.036
0.036
199
13
0.135
0.279
0.013
0.006
Zulu
Matabele
Banzabi
Moroccans
72
13
0.143
0.223
0.071
0.012
167
13
0.191
0.270
0.223
0.247
95
13
0.133
0.246
0.019
0.026
M’Zab
107
13
0.123
0.248
0.005
0.005
Merina
163
14
0.179
0.256
0.214
0.015
N: Sample size (number of individuals); n: number of alleles; Fobs: observed homozygosity; Fexp: expected homozygosity; PEW: P-value obtained by Ewens-Watterson’s test (1000 permutations);
PSL: P-value obtained by Slatkin’s test (1000 permutations)
P-values in bold are significant at the 5% level.
Table 4
Tissue Antigens 2001: 58: 211–222
219
Renquin et al : HLA class II in Aka Pygmies and Congolese
by considering only broad specificities. Alternatively, its acceptance,
in the second analysis, may be due in part to a reduced power of
Concluding remarks
the test to reject the null hypothesis when the number of alleles is
high, the sample size is low, and the selective effect is weak (41).
Thanks to the present HLA class II analyses of two new African
We should thus be care when interpreting the results of selective
population samples, the Aka Pygmies and the Bantu Congolese, and
neutrality tests applied to HLA.
the effort of previous HLA workshops to gather available data for
According to these results, balancing selection apparently influ-
many populations (23, 29, 45), a clearer picture is emerging on HLA
ences the pattern of HLA diversity in African populations, but this
diversity at the continental level of Africa. Generally speaking, the
effect is not very extensive. First, we have shown that DRB1 popula-
patterns of HLA diversity reflect population history as for most
tion genetic structure is significant and highly correlated to linguis-
classical polymorphisms, which exhibit high correlations with
tic differentiations, which indicates that population past relation-
linguistic and/or geographic differentiations (46, 47). Unlike other
ships largely influenced the pattern of HLA genetic diversity. Sec-
systems, however, a higher level of diversity than expected under a
ond, a departure from neutrality is not always demonstrated, or
neutral mode of evolution is observed in many populations, al-
demonstrable, statistically. For many samples, statistical signifi-
though to variable extents, and not at all HLA loci (37, 40, 41, 48).
cance depends on the number of alleles considered (differences be-
This diversifying selection, to which some kind of pathogen re-
tween Figs. 2 and 3). For others, we never reject the neutral hypoth-
sistance may be due (49), is expected to shift the migration-drift
esis either considering a large number of alleles or not. In the latter
balance towards the maintenance of low frequencies for a high num-
case, a possible explanation is that the effect of genetic drift
ber of alleles, and to act as an opposite force to population differen-
counterbalanced that of balancing selection, especially in Khoisan
tiation. We have indeed shown here that selection may be a possible
and Pygmies who have been isolated for a long time from other
explanation for the occurrence of some low genetic distances be-
populations. However, the Senegalese Mandenka, although being an
tween geographically or linguistically distant populations. The
endogamous community (42), probably underwent a recent popula-
overall pattern of HLA diversity is however not drastically affected
tion expansion (43), and their genetic pool may be representative of
by selective effects, and the HLA polymorphism may still be con-
broader West African genetic diversity (44).
sidered as a useful tool in anthropology.
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