HLA polymorphisms in Forros and Angolares from
Nádia Saldanhaa, Carla Spínolaa, Margarida R. Santosb, Joaquim P. Simõesb, Jácome Bruges-Armasb, António Brehma, Hélder Spínolaa
São Tomé Island
in the West coast of Africa, in the Gulf of Guinea, was discovered uninhabited
in 1470 and settled by Portuguese and people of different origins in
sub-Saharan Africa, mostly slaves recruited from the Gulf of Guinea, Congo and
Angola. During the settlement process,
sub-Saharan Africans from different geographic and ethnic origins mixed
HLA-A, HLA-B, and HLA–DRB1 loci polymorphisms were typed using
high-resolution sequence-based typing in these two ethnic groups. Allele frequencies, haplotypes and
phylogenetic analysis confirm that the West Coast of
b Institute for Molecular and Cell Biology (IBMC),
Address for correspondence: Hélder Spínola, email@example.com
human leukocyte antigen (HLA) system, the major histocompatibility
complex in humans, includes the most highly polymorphic loci in the human
genome and is located on the short arm of chromosome 6 (6p21.3), spanning over
4 Mb of
and Príncipe islands, located 300 km from the West
coast of Africa in the Gulf of Guinea as shown in Figure 1, were discovered
uninhabited by Portuguese sailors in 1470 (Peres, 1960). The archipelago was settled first by people
from different regions of sub-Saharan
the 19th century a new economic cycle based on coffee and cacao
plantations brought to the islands a new wave of sub-Saharan African people
from Cabo Verde archipelago,
people on São Tomé speak Forro, a Creole language with mixed Portuguese and Bantu
languages used by liberated slaves, known as Forros,
considered the first African inhabitants of the archipelago (Henriques, 2000; Tenreiro,
1961). Two other ethnic groups using
mixed Portuguese and African Creole languages are the Mancó,
The diverse origins of the populations of São Tomé and Príncipe archipelagos make them an interesting group in which to study genetic admixture, especially with African and European backgrounds. Previous studies on mtDNA revealed that the maternal lineages on São Tomé and Príncipe were almost completely of sub-Saharan origin, clearly belonging to a West African cluster (Mateu et al, 1997; Trovoada et al, 2004).
autosomal markers (β-globin
haplotypes, APOA1, AT3, FY, LPL,
The main aim of the present work was to analyse the HLA-A, HLA-B, and HLA-DRB1 loci allele and haplotype frequencies on present-day São Tomé and to identify European and African genetic influences. In the study we also searched for genetic differentiation between the Angolares and the Forros, the two main and most ancient ethnic groups of this archipelago.
Materials and Methods
The present study population
consisted of a total of 98 healthy unrelated males from
subjects were typed using high-resolution sequence-based typing (SBT) for HLA-A
and HLA-B according to Kurz et al. (1999) and Pozzi et al. (1999), and for HLA-DRB1 using specific
Basic genetic parameters (allele and haplotype frequencies, gene diversity, and Hardy-Weinberg equilibrium) were estimated with Arlequin v2.000 (Excoffier et al, 2005) at the three HLA loci. In the present study the Ewens-Watterson neutrality test was applied to examine the presence of selective forces influencing allelic diversity at these loci.
An analysis of molecular variance (AMOVA) was performed with Forros and Angolares groups based on Euclidean distances (Excoffier et al, 1992). Variance components were tested for significance by non parametric randomisation tests using 10,000 permutations under the null hypothesis of no population structure. The population genetic software Arlequin v2.000 was employed in all the above analyses.
Comparative analysis of the Forros and Angolares with other populations available in the literature with the same typing resolution was achieved using the software included in the PHYLIP v.3.6 software package (Felsenstein, 2004). The populations used from the literature were: Kenya, Mali, Zambia, Uganda (Cao et al, 2004), Cabo Verde, Guinea Bissau (Spínola et al, 2005c), Portugal (Spínola et al, 2005b) Madeira Island (Spínola et al, 2006) and, from the New Allele Frequency Database (Middleton, et al., 2003) web site, Italy, France, Czech Republic, India, Sudan, Cameroon, Zimbabwe (Shona), Zulu, Tunisia and Morocco. First, SEQBOOT was used to perform a bootstrap analysis from gene frequency data. The program generates multiple data sets re-sampled from the original data. Distance matrices from each replicate data set were generated using GENDIST and used as input to NEIGHBOR to produce neighbour-joining trees. A single consensus bootstrapped tree was obtained with CONSENSUS. The topology was visualized with DrawTree (Felsenstein, 2004). Principal coordinates analysis (PCO) using HLA-A, HLA-B and HLA-DRB1 allele frequencies was carried out on the MultiVariate Statistical Package MVSP3 (Kovach, 2006).
Table 1 shows HLA-A, HLA-B, and HLA-DRB1 allele frequencies in Forros and Angolares. A total of 29 and 23 HLA-A, 38 and 23 HLA-B, and 29 and 28 HLA-DRB1 alleles were found in Forros and Angolares, respectively. Forros (HLA-A 0.93, HLA-B 0.93, and HLA-DRB1 0.94) and Angolares (HLA-A 0.95, HLA-B 0.92, and HLA-DRB1 0.94) presented high values of heterozygosity and yielded non-significant results in the Ewens-Watterson neutrality test, except HLA-A in Angolares (P=0.04). Except for HLA-A in Angolares (P=0.001) and HLA-B in Forros (P=0.02), all three loci showed Hardy-Weinberg equilibrium in both groups. The exact test of population differentiation, performed by Arlequin, shows no significant differences between Forros and Angolares (P=0.37). However, all both groups are significantly different from all other populations included in the comparison (P<0.001).
most frequent HLA-A alleles found in Forros were A*0201, A*2301, and A*6802 (13% each). With a similar frequency (14%), HLA-A*6802
was also the most frequent allele in Angolares,
followed by A*2301 (9.2%). HLA*0201
tends to show lower frequencies in sub-Saharans than in Europeans (Middleton et
al., 2003), which is in agreement with frequencies found in
was the most frequent HLA-B allele in Forros (19%)
and Angolares (24%).
HLA-B*5301 is common in sub-Saharans, reaching the highest frequencies
most frequent HLA-DRB1 allele in the
The exact test of linkage disequilibrium between the three pairs of loci, an extended Fisher’s Exact Test performed with Arlequin v2.000 (Excoffier et al, 2005), was statistically significant only between HLA-A and HLA-B (P=0.016), and HLA-B and HLA-DRB1 (P=0.02) in the Forros.
The most representative three- and two-loci haplotypes with statistically significant linkage disequilibrium in the Forros and Angolares are listed in Table 2. The complete list of two- and three-loci haplotypes found in the Forros and Angolares is available the Supplementary Data file.
in Forros (3%) and A*6802-B*5301-DRB1*0804 in Angolares (4.7%) were the most frequent three-loci
haplotypes found in each group. These
two haplotypes were specific to each group and we didn’t find them in other
sub-Saharan populations, probably due to the very small number of African
populations typed on the three loci considered.
However, the related two-loci haplotype, A*6802-B*0702, was also present
second most frequent haplotype in Forros was
A*0201-B*5101-DRB1*0701 with 2.3%. This
haplotype was also found in the North of Portugal (1.1%), and in the oriental
most frequent two-loci haplotypes in Forros were
A*6802-B*5301 (5.9%), also present in
dendrogram constructed with HLA-A, HLA-B and HLA-DRB1
is shown in Figure 2a, or just with class I (HLA-A and
HLA-B) allele frequencies in Figure 2b.
These figures show a close relationship between the
and Príncipe archipelago was settled after the year
1470 by people from different origins, primarily sub-Saharan Africa, and, to a
minor extent, Europe, mostly Portuguese (Neves, 1989). The present-day population of São Tomé and Príncipe
consists of Forros (the first African inhabitants
descended from liberated slaves), Angolares (more
resistant to mixing with other groups and probably descendants of slaves who
escaped from plantations), Mancó (who live on Príncipe island) and Tonga (descendants of people who
arrived in the 19th century after slave abolition including
immigrants from Cabo Verde archipelago). Forros, Mancó and
Forros and Angolares
show no significant HLA differentiation from each other, but both groups are
significantly different from all other populations included in the comparison.
Our results are consistent with previous studies on mtDNA and
Y-chromosome that point to the West coast of
Although some references in the
literature consider the Gulf of Guinea, Congo and Angola the specific places of
origin of slaves that were brought by Portuguese to the archipelago in the
first centuries of peopling (Neves,
1989), due to the few
West African populations available for comparisons, especially
south to Gulf of Guinea,
our results only confirm West Africa as the origin of the main genetic pool of Forros and Angolares. The position of Forros
and Angolares in the dendrograms
and PCO near
lack of West African populations typed for the most polymorphic HLA loci makes
it difficult to understand the specific origin of the most frequent haplotypes
Forros are not statistically different from Angolares (P=0.03) and cluster together in phylogenetic analysis. This could mean that Angolares have a common origin with Forros, which makes plausible the hypothesis that Angolares are descendants of slaves that escaped and remained genetically isolated. In fact, founder effects, genetic drift and no admixture could explain the small differences that Angolares have with Forros on allele and haplotype frequencies. The higher European genetic input in Forros than in Angolares, as demonstrated previously on Y-chromosome studies (Gonçalves et al, 2007), could also explain the differences between them despite the hypothesis of a common origin.
previous studies (Gonçalves et al, 2007; Tomas et al,
2002; Trovoada et al, 2001) and present data, Forros and Angolares from
This work was supported by the
Portuguese Foundation for Science and Technology and the European Community
through the project Nº
The HLA haplotypes for all subjects are included in the Supplementary Data file.
New Allele Frequency Database
Arlequin Software for Population Genetics
PHYLIP: Phylogeny Inference Package
MVSP: MVSP—A Multivariate Statistical Package
Arnaiz-Villena A, Gómez-Casado E, Martínez-Laso J (2002) Population genetic relationships between Mediterranean populations determined by HLA allele distribution and a historic perspective. Tissue Antigens, 60:111-121.
Barata OS (1966) O povoamento de Cabo Verde, Guiné e São Tomé e Príncipe. In: Cabo Verde, Guiné e São Tomé e Príncipe. Curso de extensão universitária. Ano lectivo de 1965-1966. ISCSPU, Lisboa.
Boehncke WH, Loeliger C, Kuehnl P, Kalbacher H, Bohm BO, Gall H (1998) Identification of HLA-DR and -DQ alleles conferring susceptibility to pollen allergy and pollen associated food allergy. Clin Exp Allergy, 28:434-441.
Cao K, Moormann AM, Lyke KE, Masaberg C, Sumba OP, Doumbo OK, Koech D, Lancaster A, Nelson M, Meyer D, Single R, Hartzman RJ, Plowe CV, Kazura J, Mann DL, Sztein MB, Thomson G, Fernández-Vina MA (2004) Differentiation between African populations is evidenced by the diversity of alleles and haplotypes of HLA class I loci. Tissue Antigens, 63:293-325.
Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial DNA restriction data. Genetics, 131:479-491.
Garcia A (1966) A ilha de São Tomé como centro experimental do comportamento do luso nos trópicos. Separata de DTVDIA 19, Lisboa.
Gilbert M, Balandraud N, Touinssi M, Mercier P, Roudier J, Reviron D (2003) Functional Categorization of HLA-DRB1 Alleles in Rheumatoid Arthritis: The Protective Effect. Hum Immun, 64:930–935.
Gonçalves R, Jesus J, Fernandes AT, Brehm A (2002) Genetic profile of a multi-ethnic population from Guiné-Bissau (West African coast) using the new PowerPlex® 16 System kit. Forensic Sci Int, 129:78-80.
Gonçalves R, Spínola H, Brehm A (2007) Y-chromosome lineages in São Tomé e Príncipe islands: evidence of European influence. Am J Hum Biol, 19:422-8.
Henriques IC (2000) São Tomé e Príncipe—A invenção de uma sociedade. Vega e Autor.
Klein J, Sato A (2000) The HLA system; First of two parts. New Engl J Med, 343:702-709.
Kovach WL (2006) MVSP—A Multivariate Statistical Package for Windows, version 3.6. Kovach Computing Services, Pentraeth, Wales. See Web Resources.
Kurz B, Steiert
I, Heuchert G, Müller CA
(1999) New high resolution typing
strategy for HLA-A locus alleles based on dye terminator sequencing of haplotypic group-specific
Mateu E, Comas D, Calafell F, Pe A, Rez-Lezaun, Abade A, Bertranpetit J (1997)
A tale of two islands: population history and mitochondrial
Middleton D, Menchaca L, Rood H, Komerofsky R (2003) New allele frequency database. Tissue Antigens, 61:403-407. See New Allele Frequency Database under Web Resources.
Miller SA, Dykes DD, Polesky
HF (1988) A simple salting out procedure for extracting
Naik S (2003) The human HLA system. J Indian Rheumatol Assoc, 11:79-83.
Neves CA (1989) São Tomé e Príncipe na segunda metade do séc. XVIII. Colecção Memórias 2, Centro de Estudos de História do Atlântico. 1ª Edição, Funchal.
Peres D (1960) História dos descobrimentos Portugueses. Coimbra. 2ª Edição.
Pozzi S, Longo A, Ferrara GB (1999) HLA-B locus sequence-based typing. Tissue Antigens, 53:275-281.
Riley E, Olerup O (1992) HLA polymorphism and evolution. Immunol Today, 13:333-335.
Romana H (1997) São Tomé e Príncipe: Elementos para uma análise antropológica das suas vulnerabilidades e potencialidades. Instituto Superior de Ciência Sociais e Políticas. Universidade Técnica de Lisboa, Lisboa.
Rosa A, Brehm A, Kivisild T, Metspalu E, Villems R (2004) MtDNA profile of West Africa Guineans: Towards a better understanding of the Senegambia region. Ann Hum Genet, 68:340-352.
Rosa A, Ornelas C, Brehm A, Villems R (2006) Population data on 11 Y-chromosome STRs from Guiné-Bissau. Forensic Sci Int, 157:210-217.
Sanchez-Mazas A (2001) African diversity from the HLA point of view: Influence of genetic drift, geography, linguistics, and natural selection. Hum Immunol, 62:937-948.
Seibert G (1998) A Questão da Origem dos Angolares de São Tomé. Brief Papers, 5/98. CESA, Lisboa.
Spínola H, Brehm A, Williams F, Jesus J, Middleton D (2002) Distribution of HLA alleles in Portugal and Cabo Verde: Relationships with the slave trade route. Ann Hum Genet, 66:285-296.
Spínola H, Brehm A, Bettencourt B, Middleton D, Bruges-Armas J (2005a) HLA class I and II polymorphisms in Azores show different settlements in Oriental and Central Islands. Tissue Antigens, 66:217-230.
Spínola H, Middleton D, Brehm A (2005b) HLA genes in Portugal inferred from sequence-based typing: in the crossroad between Europe and Africa. Tissue Antigens, 66:26-36.
Spínola H, Bruges-Armas J, Middleton D, Brehm A (2005c) HLA polymorphisms in Cabo Verde and Guiné-Bissau inferred from sequence-based typing. Hum Immunol, 66:1082–1092.
Spínola H, Bruges-Armas J, Mora MG, Middleton D, Brehm A (2006) HLA genes in Madeira Island (Portugal) inferred from sequence-based typing: Footprints from different origins. Hum Immunol, 43:1726-1728.
Tenreiro F (1961) A ilha de São Tomé; Memórias da Junta de Investigação do Ultramar. 2ª Edição 24, Lisboa.
Tomas G, Seco L, Seixas S, Faustino P, Lavinha J, Rocha J (2002) The peopling of Sao Tome (Gulf of Guinea): origins of slave settlers and admixture with the Portuguese. Hum Biol, 74:397-411.
MJ, Alves C, Gusmão L, Abade A, Amorim A, Prata MJ, (2001) Evidence for population sub-structuring in
São Tomé e Príncipe as inferred from Y-chromosome
Trovoada MJ, Pereira L, Gusmão L, Abade A, Amorim A, Prata MJ (2004) Pattern of mtDNA variation in three populations from São Tome e Principe. Ann Hum Genet, 68:40-54.
Williams F, Meenagh A, Single, R, McNally M, Kelly P, Nelson M, Meyer D, Lancaster A, Thomson G, Middleton D (2004) High resolution HLA-DRB1 identification of a Caucasian population. Hum Immun, 65:66-77.