doi: 10.1111/iji.12116
HLA-DQA1 and HLA-DQB1 genes in Tsachilas Indians from Ecuador: new insights in population analysis by Human Leukocyte Antigens A. Iorio*,†, F. De Angelis*, A. Garzoli*, A. Battistini* & G. F. De Stefano*
Summary Human Leucocyte Antigen (HLA) loci are widely known for their role in the generation of immune responses and are often considered to be effective in reconstructing human relationships. This is due to the high degree of polymorphism and the rarity of recombination observed at HLA loci. In this study, we have made an attempt to support the potential of HLA class II loci by analysing DQA1 and DQB1 in 52 Ecuadorians with ties to the Tsachilas community. Little is known about this populations either ethnologically or historically: they are considered retaining much of the ancient Chibchan culture in spite of the lack of significant genetic characterization. A total of 21 alleles were observed, with very low heterozygosity. The obtained data were then assessed for relationship reconstruction. The compiled database of 63 populations was segregated and resolved in clusters corresponding to the ethnogeographic distribution of the populations. This analysis of Central and Southern Amerindians allowed us to support a historical hypothesis related to the origin and migration of Ecuadorian people. Indeed, the relationships with neighbour human groups, especially Cayapas and Colombians, could shed light on the genetic similarity within ancient Chibchan culture that was dispersed by tribes coming up the Barbacoas. This indicates that if an appropriate analysis was to be carried out on a set of populations representative of different geographic locations, and that analysis was properly interpreted, then there would be a high possibility that HLA class II loci could infer accurate assessments, as revealed by uniparental markers.
* Department of Biology, University of Rome Tor Vergata, Rome, Italy and † Clinical Pathophysiology Center, AFaR, “San Giovanni Calibita” Fatebenefratelli Hospital, Rome, Italy Received 11 November 2013; revised 11 February 2014; accepted 23 February 2014 Correspondence: Flavio De Angelis, Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy. Tel: + 390672594350; Fax: + 39062023500; E-mail: fl[email protected]
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Introduction In recent decades, new contributions in population genetics and molecular anthropology have allowed us to better define the extent of genetic variability in humans, as well as the processes that underlie it (Cavalli-Sforza & Feldman, 2003). Today, human DNA is known to be the main object of the population studies, yet the choice of populations, markers, genotyping procedures and statistical methods play a key role in these analyses too (Jorde & Wooding, 2004; CavalliSforza, 2005). The preferred regions examined in population studies tend to exhibit a high degree of polymorphism, have significantly different frequency distributions in different groups and carry signature haplotypes in different populations (Cavalli-Sforza, 2005). Several polymorphic regions of the human genome have been used in recent years to infer population relationships, and, among them, our attention is focused on the Human Leucocyte Antigen (HLA) genomic area. The HLA complex is located in three main regions of chromosome 6. These include the class I, class III (telomeric) and class II (centromeric) loci (Middleton, 1999). Among class II loci, our interest is focused on HLA-DQ; like every HLA class II, it encodes proteins on specific antigen-presenting cells. These molecules are heterodimers encoded by DQA1 and DQB1 genes. HLA genetic variation has come under recent inquiry because of its extreme polymorphic nature (Ping & Wang, 2003). The high rate of polymorphism of HLA loci offers us the possibility to infer human population relationships: evidence suggests that HLA class II loci are comparable to mtDNA and Y chromosome markers in phylogenetic assessment (Arnaiz-Villena et al., 2005; Bharadwaj et al., 2007). They are both vastly polymorphic and highly linked loci that result in a unique linkage disequilibrium pattern, signature alleles and haplotype segregation among different populations. The actual preference of lineage-based markers (NRY and mtDNA) is mainly due to their nonrecombining property, but only maternal or paternal lineages do not represent the whole genetic make-up of an individual. Therefore, a biparentally segregated marker, such as HLA class II loci, could be a useful candidate
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for inferring relationships between different human populations (Cavalli-Sforza & Feldman, 2003; Bharadwaj et al., 2007); this approach was employed to analyse the relationships of an Ecuadorian ethnic group among worldwide populations. The actual cultural diversity of the Ecuadorian country was represented by 17 main ethnic groups and comes from several processes of adaptation to a wide range of habitats, immigration, the Spanish conquest in the sixteenth century, and the arrival and the mixture of Africans (De la Torre & Balslev, 2008). According to the 2001 census, the Ecuadorian population is composed mostly of mestizos, followed by people of European origin and indigenous Amerindians. Social studies conclude that the Ecuadorian indigenous population ranges between 8% and 12%, and spans all provinces of Ecuador (Guerrero, 2005). The Ecuadorian population may be subdivided according to criteria of self-identification and spoken language. The multi-ethnicity of Ecuador has already been accepted in its own constitution; indigenous languages are recognized as part of the cultural heritage of the country (Trujillo, 1992). There are 14 spoken languages throughout diverse cultures and beyond the Spanish, which comes from the Indo-European language family, and Wao Tededo and El a’ingae, which are isolated languages (De la Torre & Balslev, 2008), and the rest are grouped in six Amerindian linguistic families: Barbacoa, Choc o, Quechua, Zapara, Aents and Western Tucano. The Barbacoa, whose diversity dates back approximately 3300 years (De la Torre & Balslev, 2008), is comprised of these languages: Awapit, Chafi’ki and Tsafi’ki of Ecuador, as well as Guambiano, Totoro and Barbacoa of Colombia (De Stefano, 1994; Curnow & Liddicoat, 1998). The Tsafi’ki language is still spoken by an indigenous population known as the Tsachilas or Colorados. They live in the Pichincha province, at the Canton of Santo Domingo de los Colorados (Figure S1). Little is known about Tsachilas either ethnologically or historically. Karsten (Karsten, 1988) stated Tsachilas, Cayapas and Cuna of Colombia retain much of the ancient Chibchan culture, which originally spread throughout all what now is Colombia. The languages of these populations show the same basic etymological origins, yet even the separation among them has brought variants in pronunciation (Von Hagen, 1988). The ancient Chibchan culture was subsequently dispersed by tribes coming up the actual Barbacoas, which assimilated the populations. The Tsachilas constitute approximately 2500 people clustered in eight communities. Hunting, fishing and gathering were originally their main activities; however, these activities are no longer practiced, due to a lack of suitable land. Agriculture is still important, but the marketing of agriculture dominates. Currently, they participate in the market economy through livestock, marketing, ecotourism and the practice of traditional medicine. Inbreeding is allowed, and practices within their groups retain community
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 222–230
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traditions, rituals and even their dress. Men manage their hair by bundling it into a cap painted with achiote (Bixa orellana) and oil: this is the reason for the nickname ‘Colorados’. As noted, almost nothing of their traditional territory remains: today, Santo Domingo de los Colorados is an economic hub between the coast and the Sierra, and it represents a collection and marketing centre of a large number of products, so the Tsachilas have been absorbed into the traditional economy of Ecuador (Barfod & Kvist, 1996). This research aimed to analyse the HLA-DQ profile of this population to ascertain its genetic relationships with neighbour groups. Several studies of HLA class I and class II variation in Amerindian populations have been carried out for anthropological as well as evolutionary and clinical studies (Tsuneto et al., 2003; Parolin & Carnese, 2009; Arnaiz-Villena et al., 2010; Vargas-Alarcon et al., 2010; De Angelis et al., 2012). These data were compared with other Amerindian groups and worldwide populations to clarify the relationships among northern inhabitants of South America and to highlight the power of HLA system in determining population relationships.
Materials and method Population sample
This study explores the allele composition of DQA1 and DQB1 genes in the Tsachilas population of the Chiguilpe community. The sample pertains to 52 unrelated healthy individuals; they were blood donors who volunteered for this study after informed consent. Recruiting was carried out in the Chiguilpe area by one of us (GFDS), in accordance with ethical permission standards. DNA was extracted from whole blood using standard protocols (Miller et al., 1998) and was stored at 80°C. Tsachilas samples were compared with other target populations to obtain a pattern of genetic distances useful to test the quality of HLA to ascertain the human relationships and to demonstrate that the analysis of HLA loci is a valuable tool in anthropological studies. The 62 selected populations are shown in Table 1. The choice of populations was guided by the availability of frequency data both for HLA-DQA1 and HLA-DQB1 in the open-source database on www.allelefrequencies.net (Gonzalez-Galarza et al., 2011), that allowed to analyse approximately 11.742 chromosomes. Every population was previously evaluated by anthropological study, and no case–control studies have been included in the analysis to avoid an altered distribution of allelic variants. HLA typing
HLA-DQA1 and HLA-DQB1 loci were high-resolution typed by direct sequencing of exons 2 and 3. The
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Table 1. Reference populations from www.allelefrequencies.net ID
Population
Acronyms
Location
Sample size
References
1 2 3 4 5 6 7 8 9 10 11 12 13
Amhara Bamileke Cameroon Cameroon Saa Congo Kinshasa Bantu Equatorial guinea Bioko Island Bubi Gabon Haut-Ogooue Dienga Gambia Kenya Morocco Oromo Uganda Baganda Zimbabwe Harare Shona
Amh Bam Cam CamS Congo EquaGBB GabonHOD Gambia Ken Mor Oromo Uga Zim
Africa
46 34 126 172 90 100 167 146 144 96 61 47 230
Unpublished Unpublished (Gonzalez-Galarza et al., 2011) (Begovich et al., 2001) (Renquin et al., 2001) (de Pablo et al., 1997) (Migot-Nabias et al., 1999) (Begovich et al., 2001) (Luo, 2006) (Gomez-Casado et al., 2000) Unpublished Begovich et al., 2001; (Louie, 2006)
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Afroecuadorians Argentina Chiriguanos Argentina Gran Chaco Eastern Toba Argentina Gran Chaco Mataco Wichi Argentina Gran Chaco Western Toba Brazil Central Plateau Xavantes Brazil Guaranı Kaiowa Brazil Guarani M Bya Cayapas Colombia Amazonas Coreguaje Colombia Arhuaco Sierra Nevada Colombia East Amazon Region Nukak Colombia Guajira Peninsula Wayuu Colombia NE plains Sikuani Colombia NW Choco Colombia Providencia Island Africans Colombia S.N. de S.ta Marta Arhuaco Costa Rica and Panama Mexico Sonora Seri Paraguay Ache Quechua Peru Venezuela Sierra de Perija Yucpa
Afroec ArgChi ArgGCET ArgGCMW ArgGCWT BraCPX BraGK BraGMB Cay ColAC ColASN ColEARN ColGPW ColNEPS ColNWC ColPIA ColSMA CosPA MexSS ParA PerQ VenS
America
75 54 135 49 19 74 155 93 74 30 107 20 88 27 20 30 107 56 34 87 44 73
(De Angelis et al., 2012) (Gonzalez-Galarza et al., 2011) (Cerna et al., 1993) (Cerna et al., 1993) (Cerna et al., 1993) (Cerna et al., 1993) (Tsuneto et al., 2003) (Tsuneto et al., 2003) (De Angelis et al., 2012) (Trachtenberg et al., 1996) (Yunis et al., 1994) (Trachtenberg et al., 1996) (Yunis et al., 1994) (Trachtenberg et al., 1996) (Trachtenberg et al., 1996) (Trachtenberg et al., 1996) (Yunis et al., 1994) (Erlich, 2006) (Gorodezky, 2006) (Tsuneto et al., 2003) (Tsuneto et al., 2003) (Layrisse et al., 2001)
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Australia Aborigens Cape York Australia Aborigens Kimberly China Beijing and Xian China Canton Han Cook Island Rarotonga Fiji Viti Levu India Bombay India NE kayastha India Uttar Pradesh Hindu Indonesia Java Yogyakarta region Iran Azeris Iran Baloch Iran Kurds Israel Arab Israel Ashkenazi Israel ethiopian jews Japan
AusACY AusAK ChiBX ChiCH CookRar FijiVL IndB IndNEK IndUPH IndoJYR IranA Iranb IranK IsrA IsraA IsraEJ Jap
Asia
103 41 171 264 78 57 59 190 202 62 100 100 100 109 80 122 50
(Gao, 2000) (Gao, 2000) (Gonzalez-Galarza et al., 2011) (Trachtenberg et al., 2007) (Gao et al., 1992) (Gao et al., 1992) (Begovich et al., 2001) (Agrawal et al., 2008) (Gonzalez-Galarza et al., 2011) (Gao et al., 1992) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Martinez-Laso et al., 1996) (Gonzalez-Galarza et al., 2011) (Geng et al., 1995)
53 54 55 56 57 58 59 60 61 62
Croatia Gorski Kotar Croatia Krk Island Croatia Krk island Dobrinj Czech Gipsy Czech Republic Denmark England Georgia Svaneti Region Svan Greece Crete Spain North Cantabria
Crogk CroKID CroKID CzeG Cze Den Eng Georg GreC SpaNC
Europe
63 212 28 34 106 55 177 80 135 83
(Martinovic et al., 1997) (Gonzalez-Galarza et al., 2011) (Martinovic et al., 1997) (Fernandez-Vina et al., 1992) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Sanchez-Velasco & Leyva-Cobian, 2001) (Arnaiz-Villena et al., 1999) (Gonzalez-Galarza et al., 2011)
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choice for these regions was due to their crucial role in encoding the HLA molecules; in fact, these exons, respectively, encode for the a1 and b1 regions of the HLA-DQ subunits, which are the regions that form the antigen cleft. The primers for amplification and sequencing are listed in Table S1; they were used according to references (Cordovado et al., 2005; van Dijk et al., 2007; De Angelis et al., 2012). Conditions for the polymerase chain reaction (PCR) assay by custom-designed primers were as follows: 10 min at 95°C, 35 cycles of 30 s at 95°C, 1 min at 53.5°C, 30 s at 72°C and a final extension of 7 min at 72°C. The sequencing reactions used BigDye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions and amplification forward primers. All the sequences were compared to the IMGT/HLA Database v.3.5.0 (Robinson et al., 2000) to determine the allelic assignments at the two HLA class II loci. All class II alleles included in the present study are four-digit resolution data. Statistical analysis
Overall distributions of HLA-DQ alleles were determined by the direct counting method. Deviation from the Hardy–Weinberg equilibrium was examined with Arlequin software, version 3.5.1.2 (Excoffier et al., 2005), with the default setting, where the exact P-value is calculated through the Markov chain method (Guo & Thompson, 1992). HLA-DQA1 and HLA-DQB1 haplotypes have been estimated by Phase v. 2.1 (Stephens et al., 2001). To infer relationships between different populations by HLA class II loci, a database of 62 geographically and ethnically different populations has been compiled. Similarity reconstruction with statistical bootstrap involving 1000 replicates was carried out by Nei’s chord distances (Nei, 1987) and neighbour-joining algorithm (NJ), using the PHYLIP v 3.69 software package (Felsenstein, 2009). Genetics distances were also used to draw up a multivariate display of population similarity through multidimensional scaling (MDS). The multivariate analysis reduces multidimensional space to two/three dimensions graphically represented, with minimal loss of information.
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Results and discussion Table 2 shows the HLA-DQA1 and HLA-DQB1 allele frequencies in the Tsachilas population. This points out an expected heterogeneity that encompasses 11 variants at locus HLA-DQA1, with the leading frequency of three alleles, HLA-DQA1*0301 with 15% (n = 16), *0401 with 29% (n = 30) and *0501 with 28% (n = 29), while the other allelic variants are somewhat less represented. The same heterogeneity is shown by locus HLADQB1; in fact, the number of alleles is quite the same, but the frequency distribution is less skewed. The leading presence of HLA-DQB1*0201 (16%) is due to 17 alleles, followed by *0301 with 21 alleles (20%) and the *0302 with 24 alleles (23%) (Table 2). The observed allele frequencies of the HLA-DQA1 and HLA-DQB1 were compared to the theoretically expected frequencies and significant deviations from the Hardy–Weinberg equilibrium and were observed especially for the HLA-DQA1 locus (DQA1: P = 0.00000; DQB1: P = 0.00005). It is interesting to note the high frequency of HLADQA1*0401 in the sample that might be evidence of putative genetic protection against helminth infections, in particular onchocerciasis, although no individual data are available about the presence/absence of disease (De Angelis et al., 2012). The establishment of a new, increasing focus on onchocerciasis was suspected in the Tsachilas because of the immigration of Cayapas Amerindians from hyperendemic onchocerciasis focus over the last 20 years (Cooper et al., 2001) and the presence of the vector species Simulium exiguum (Charalambous et al., 1997). The random sampling of apparently healthy people might be inclusive of people protected against onchocerciasis carrying the protective allele DQA1*0401. No haplotype is markedly frequent in the sample, as reported in Table S2. These findings highlight the absence of an evident linkage disequilibrium pattern in the Amerindian sample. To test the Tsachilas relationships with other human groups, the distribution of HLA-DQ alleles was compared to other populations by MDS and NJ dendrogram. The population choice was arbitrarily carried out on the basis of geographical distribution and
Table 2. DQA1 and DQB1 allele frequencies in Tsachilas DQA1
*0102
*0201
*0301
*0302
*0303
*0401
*0402
*0501
*0502
*0503
*0601
TOT
N alleles % S.E.
4 3.8 0.019
5 4.8 0.021
16 15.4 0.035
3 2.9 0.016
2 1.9 0.013
30 28.8 0.044
5 4.8 0.021
29 27.9 0.044
3 2.9 0.016
1 1 0.009
6 5.8 0.023
104 100
DQB1
*0201
*0202
*0301
*0302
*0303
*0304
*0401
*0402
*0501
*0502
TOT
N alleles % S.E.
17 16.3 0.036
6 5.8 0.023
21 20.2 0.039
24 23.1 0.041
9 8.7 0.028
2 1.9 0.013
8 7.7 0.026
2 1.9 0.013
8 7.7 0.026
7 6.7 0.024
104 100
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depending on the availability of comparable data for both analysed loci. The analyses were separately carried on for each locus (HLA-DQA1 and HLA-DQB1) and then used by the pooled loci (HLADQA1 + HLA-DQB1). Despite some inconsistencies probably attributable to the limited number of markers, the MDS made on data from the DQA1 locus reveals the presence of four main clusters: roughly due to ethnogeographic groups of Asians, South Americans, Europeans and Africans (Figure S2). Indeed, the Amerindians are well clustered at the edge of the distribution by Dimension 1, with variable eigenvalues by Dimension 2 and Dimension 3. Tsachilas are placed in the middle, suggesting a genetic contribution by both Central Americans (Costa Rica and Panama; Mexico) and northern South American human groups (Colombian Amerindians; Brazilians). The clustering of the other ethnogeographic groups is less defined than Amerindians. Further discussion can be made about the similarity between the African peoples and Afroecuadorians, presumably due to the genetic origin of the African communities of Ecuador (De Stefano, 1994; MartinezLabarga et al., 1999), as the result of the settlement of human groups conducted in the Americas by the slave trades from Guinea Gulf in the seventeenth century (De Stefano, 1994). The MDS for the HLA-DQB1 (Figure S3) locus shows that the ethnogeographical differences are less defined, probably due to selective convergence; the clear scenario has been replaced by a more nuanced situation, and the Tsachilas sample is located at the edge of American group. This could be due to functional constraints of the peptide encoded by HLA-DQB1: the section of the HLA-DQ heterodimer essential to the housing of the antigenic peptide within the binding cleft of the molecule (Menconi et al., 2010). Thus, it could be estimated how this locus might be subjected to a wider selective force, resulting in a loss of quality in the discriminatory inference of population relationships. The NJ dendrograms made by standard Nei genetic distances on both loci and 1.000.000 permutations are consistent with MDS representations. The MDS of both the loci highlights, once again, the presence of four different clusters, with better resolution; the improvement is a consequence of both loci having been considered (Figure 1). The graph reveals the remarkable diversity of the American population, which is made up of different ethnic groups (De Stefano, 1994; Polimanti et al., 2011). This is also highlighted in Figure 2, in which only new world groups are compared. The MDS plot highlights (stress = 0.12) a clear differentiation among populations that might be related to the origin of each human group. Tsachilas lie in the right upper quadrant, along with Central Amerindians and northern South Amerindians, and this relatedness may be a detector of their linguistic similarity.
Figure 1. Tridimensional multidimensional scaling (MDS) HLADQA1+HLA-DQB1; Stress = 0.14. Black dots are Africans, white dots are South Americans, black squares are Asians and white squares are Europeans. Tsachilas sample is marked by a black star.
First dimension shows a cluster for Argentinians and Brazilians that refers to people living around Chaco region, that is a central South American area boundaried by Southern Brazil, Northern Argentina, Bolivia and Paraguay. Nowadays, this territory is largely uninhabited except for different hunter-gatherer peoples, making the area a great cultural gathering and genetic exchange (Cabana et al., 2006). Colombians are more scattered in the plot because of their peculiar genetic background. This scenario has already been suggested by mitochondrial DNA studies showing asymmetrical Amerindian admixture in Colombia. Amerindians should experience weak matrilineal admixture with Caucasians or Africans that reached the area, resulting in different patterns of past ethnic admixture in the geographic region now encompassing Colombia, which is also reflected in much of the region’s cultural diversity (Rodas et al., 2003). The tight genetic proximity between Tsachilas and Costa Rica/Panama sample is worthy of consideration: these areas are considered the centre of Chibchan languages (Greenberg, 1987). The reported Central Amerindians are linguistically clustered in the Macro-Chibchan family (Rhulen, 1991), while the Tsachilas are a Barbacoan-derived people from the ancient Yumbos human group (Lippi & Gudi~ no, 2004). According to archaeological research, the Yumbos occupied southwestern Colombia and northeastern Ecuador. Less than 10% linguistic correspondence between Macro-Chibchan and Barbacoa languages was detected, and doubts on the rightness of the Macro-Chibchan group have been advanced (Wheeler, 1972; Constenla, 1991). It could be hypothesized that Barbacoan might be a mix of Chibchan language and languages from the Andes. On the basis of this hypothesis, it has been proposed that the Chibchan populations moved southwards and mixed with
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Figure 2. Bidimensional multidimensional scaling (MDS) HLA-DQA1+HLA-DQB1 of Central Amerindians samples; Stress = 0.12.
Andeans (Lippi, 2003). The genetic affinity between Tsachilas and Costa Rican sample might account this hypothesis: Tsachilas could represent a Chibchan group southwards moving with weaker genetic admixing than other Chibchan-derived people as the Cayapas Amerindians are (Von Hagen, 1988). Archaeological data confirm that Barbacoans split from the Macro-Chibchan southward from Central Colombia, and they reached south Colombia, northern Ecuador and the western slope of the Andes. Therefore, Barbacoans represent southern populations closer to Chibchan, and they probably mixed with Andean human groups. Moreover, according to these data, Lippi proposed the ancient Yumbos derived from the Caras, that is, an Ecuadorian highplane, prehispanic population (Lippi, 2003). Some Spanish reports identified South and North Yumbos: North Yumbos would seem to have derived from Caras and afterwards would have reached Western Pichincha, according to ‘Toras’ (archaeological buildings). The above results may support the hypothesis suggested by some authors (Melton et al., 2007), concerning an expansion of the Chibchan-speaker groups from the central to southern portion of the continent. Subsequently, the phenomena of genetic drift would produce the considerable genetic diversity that is observed in allele frequencies among different populations. While archaeological and linguistic research has found a cultural association between Central and South American Chibchan-speaking populations, the biological relationship has long remained largely unresolved. This study demonstrated that northern South and lower Central American Chibchan speakers might share a similar genetic structure, but if they share a common ancestry it occurred in the distant past.
Conclusions Several polymorphic regions of the human genome have been used in recent years to determine human
© 2014 John Wiley & Sons Ltd International Journal of Immunogenetics, 2014, 41, 222–230
population relationships and, like all the preferred regions that exhibit a high degree of polymorphism, the HLA loci could be used for this aim. Overall, the analysis of the two HLA class II loci has depicted a geo-ethnic distribution indicating that the presence of numerous alleles (against biallelic SNPs, mtDNA and Y chromosome) does not interfere with the phylogenetic information content of the loci, provided that the frequency distribution of the populations is significantly different. Instead, it increases the chance of the presence of signature alleles and specific haplotypes in cases of closely linked loci, like HLA. The present study has allowed us to demonstrate the effective discriminatory potential of the two HLA loci; in fact, the results show that the clusters might reflect the origin and the geographical distribution of the populations, highlighting also genetic relationships that could be modified by historical and cultural events such as migration or admixture. Indeed, HLA analysis might be an aid in population analysis, achieving similar results than mtDNA. However, there are potential problems associated with the use of HLA loci in inferring human population relationships. Most important is the nonuniformity of HLA typing methodologies, as some studies are based on low-resolution typing while others are based on high-resolution typing. Preferably, if all the HLA loci (both class I and class II) are analysed with a high resolution of all the alleles and a proper statistical interpretation based on more logistic approaches is carried out, then the HLA loci can be an ideal marker to infer genetic differences between inter- and intra-geo-ethnic groups. Moreover, if the property of rarity of the recombination at the HLA loci is fully explored to assign extended haplotypes, then there will be a high possibility that the HLA loci could reconstruct the human phylogenies as exactly and as accurately as those deciphered by the mtDNA and Y chromosome markers. This attempt may be useful to develop a new aspect of the HLA studies,
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although the disease association ones are, to date, the leading fieldworks in HLA genetics.
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Renquin, J., Sanchez-Mazas, A., Halle, L., Rivalland, S., Jaeger, G., Mbayo, K., Bianchi, F. & Kaplan, C. (2001) HLA class II polymorphism in Aka pygmies and Bantu congolese and a reassessment of HLA-DRB1 african diversity. Tissue Antigens, 58, 211. Rhulen, M.. (1991) A Guide to the World’s Languages. Volume 1: Classification. Stanford University Press, Stanford. Robinson, J., Malik, A., Parham, P., Bodmer, J.G. & Marsh, S.G. (2000) Imgt/hla database–a sequence database for the human major histocompatibility complex. Tissue Antigens, 55, 280. Rodas, C., Gelvez, N. & Keyeux, G. (2003) Mitochondrial DNA studies show asymmetrical amerindian admixture in afrocolombian and mestizo populations. Human Biology, 75, 13. Sanchez-Velasco, P. & Leyva-Cobian, F. (2001) The HLA class I and class II allele frequencies studied at the DNA level in the Svanetian population (upper Caucasus) and their relationships to western european populations. Tissue Antigens, 58, 223. Stephens, M., Smith, N. & Donnelly, P. (2001) A new statistical method for haplotype reconstruction from population data. American Journal of Human Genetics, 68, 978. Trachtenberg, E.A., Keyeux, G., Bernal, J., Noble, J.A. & Erlich, H.A. (1996) Results of expedicion humana. Ii. Analysis of HLA class ii alleles in three african american populations from colombia using the pcr/ssop: identification of a novel dqb1*02 (*0203) allele. Tissue Antigens, 48, 192. Trachtenberg, E., Vinson, M., Hayes, E., Hsu, Y.M., Houtchens, K., Erlich, H., Klitz, W., Hsia, Y. & Hollenbach, J. (2007) HLA class I (A, B, C,) and class II (DRB1, DQA1, DQB1, DPB1) alleles and haplotypes in the Han from southern China. Tissue Antigens, 70, 455. Trujillo, O.V. (1992) Guadalupe: conflict and civil rights in a tricultural setting. In: Martin Luther King: Living the Dream (ed. G. Bataille). Arizona State University, Tempe, AZ, USA. Tsuneto, L.T., Probst, C.M., Hutz, M.H., Salzano, F.M., Rodriguez-Delfin, L.A., Zago, M.A., Hill, K., Hurtado, A.M., Ribeiro-dos-Santos, A.K. & Petzl-Erler, M.L. (2003) Hla class ii diversity in seven amerindian populations. Clues about the origins of the ache. Tissue Antigens, 62, 512. Vargas-Alarcon, G., Granados, J., Rodriguez-Perez, J.M., Parga, C., Perez-Hernandez, N., Rey, D., Zuniga, J. & ArnaizVillena, A. (2010) Distribution of hla class ii alleles and haplotypes in mexican mestizo population: comparison with other populations. Immunological Investigations, 39, 268. Von Hagen, W. (1988) Los indios tsachela del oeste ecuatoriano. In: Los Cl asicos De La Etnografıa Sobre Los Colorados (ed. J. Juncosa). Abya-yala, Quito. Wheeler, A. (1972) Proto-chibchan. In: Comparative Studies in Amerindian Languages (ed. E. Matteson). The Hague, Mouton. Yunis, J.J., Ossa, H., Salazar, M., Delgado, M.B., Deulofeut, R., de la Hoz, A., Bing, D.H., Ramos, O., Yunis, E.J. & Yunis, E.J. (1994) Major histocompatibility complex class II alleles and haplotypes and blood groups of four amerindian tribes of northern Colombia. Human Immunology, 41, 248.
Supporting Information Additional supporting information may be found in the online version of this article: Table S1 PCR and sequencing primers. Table S2 Haplotype HLA-DQA1 and HLA-DQB1 in Tsachilas. Figure S1 Geographical location of Canton de Santo Domingo de los Colorados.
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Figure S2 Tridimensional MDS for HLA-DQA1, based on Nei’s chord distances; Stress = 0.13. Black dots are Africans, white dots are South Americans, black squares are Asians and white squares are Europeans. Tsachilas sample is marked by a black star.
Figure S3 Tridimensional MDS HLA-DQB1 based on Nei’s chord distances; Stress = 0.12. Black dots are Africans, white dots are South Americans, black squares are Asians and white squares are Europeans. Tsachilas sample is marked by a black star.
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HLA-DQA1 and HLA-DQB1 genes in Tsachilas Indians from Ecuador: new insights in population analysis by Human Leukocyte Antigens A. Iorio*,†, F. De Angelis*, A. Garzoli*, A. Battistini* & G. F. De Stefano*
Summary Human Leucocyte Antigen (HLA) loci are widely known for their role in the generation of immune responses and are often considered to be effective in reconstructing human relationships. This is due to the high degree of polymorphism and the rarity of recombination observed at HLA loci. In this study, we have made an attempt to support the potential of HLA class II loci by analysing DQA1 and DQB1 in 52 Ecuadorians with ties to the Tsachilas community. Little is known about this populations either ethnologically or historically: they are considered retaining much of the ancient Chibchan culture in spite of the lack of significant genetic characterization. A total of 21 alleles were observed, with very low heterozygosity. The obtained data were then assessed for relationship reconstruction. The compiled database of 63 populations was segregated and resolved in clusters corresponding to the ethnogeographic distribution of the populations. This analysis of Central and Southern Amerindians allowed us to support a historical hypothesis related to the origin and migration of Ecuadorian people. Indeed, the relationships with neighbour human groups, especially Cayapas and Colombians, could shed light on the genetic similarity within ancient Chibchan culture that was dispersed by tribes coming up the Barbacoas. This indicates that if an appropriate analysis was to be carried out on a set of populations representative of different geographic locations, and that analysis was properly interpreted, then there would be a high possibility that HLA class II loci could infer accurate assessments, as revealed by uniparental markers.
* Department of Biology, University of Rome Tor Vergata, Rome, Italy and † Clinical Pathophysiology Center, AFaR, “San Giovanni Calibita” Fatebenefratelli Hospital, Rome, Italy Received 11 November 2013; revised 11 February 2014; accepted 23 February 2014 Correspondence: Flavio De Angelis, Department of Biology, University of Rome Tor Vergata, Via della Ricerca Scientifica 1, 00133, Rome, Italy. Tel: + 390672594350; Fax: + 39062023500; E-mail: fl[email protected]
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Introduction In recent decades, new contributions in population genetics and molecular anthropology have allowed us to better define the extent of genetic variability in humans, as well as the processes that underlie it (Cavalli-Sforza & Feldman, 2003). Today, human DNA is known to be the main object of the population studies, yet the choice of populations, markers, genotyping procedures and statistical methods play a key role in these analyses too (Jorde & Wooding, 2004; CavalliSforza, 2005). The preferred regions examined in population studies tend to exhibit a high degree of polymorphism, have significantly different frequency distributions in different groups and carry signature haplotypes in different populations (Cavalli-Sforza, 2005). Several polymorphic regions of the human genome have been used in recent years to infer population relationships, and, among them, our attention is focused on the Human Leucocyte Antigen (HLA) genomic area. The HLA complex is located in three main regions of chromosome 6. These include the class I, class III (telomeric) and class II (centromeric) loci (Middleton, 1999). Among class II loci, our interest is focused on HLA-DQ; like every HLA class II, it encodes proteins on specific antigen-presenting cells. These molecules are heterodimers encoded by DQA1 and DQB1 genes. HLA genetic variation has come under recent inquiry because of its extreme polymorphic nature (Ping & Wang, 2003). The high rate of polymorphism of HLA loci offers us the possibility to infer human population relationships: evidence suggests that HLA class II loci are comparable to mtDNA and Y chromosome markers in phylogenetic assessment (Arnaiz-Villena et al., 2005; Bharadwaj et al., 2007). They are both vastly polymorphic and highly linked loci that result in a unique linkage disequilibrium pattern, signature alleles and haplotype segregation among different populations. The actual preference of lineage-based markers (NRY and mtDNA) is mainly due to their nonrecombining property, but only maternal or paternal lineages do not represent the whole genetic make-up of an individual. Therefore, a biparentally segregated marker, such as HLA class II loci, could be a useful candidate
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for inferring relationships between different human populations (Cavalli-Sforza & Feldman, 2003; Bharadwaj et al., 2007); this approach was employed to analyse the relationships of an Ecuadorian ethnic group among worldwide populations. The actual cultural diversity of the Ecuadorian country was represented by 17 main ethnic groups and comes from several processes of adaptation to a wide range of habitats, immigration, the Spanish conquest in the sixteenth century, and the arrival and the mixture of Africans (De la Torre & Balslev, 2008). According to the 2001 census, the Ecuadorian population is composed mostly of mestizos, followed by people of European origin and indigenous Amerindians. Social studies conclude that the Ecuadorian indigenous population ranges between 8% and 12%, and spans all provinces of Ecuador (Guerrero, 2005). The Ecuadorian population may be subdivided according to criteria of self-identification and spoken language. The multi-ethnicity of Ecuador has already been accepted in its own constitution; indigenous languages are recognized as part of the cultural heritage of the country (Trujillo, 1992). There are 14 spoken languages throughout diverse cultures and beyond the Spanish, which comes from the Indo-European language family, and Wao Tededo and El a’ingae, which are isolated languages (De la Torre & Balslev, 2008), and the rest are grouped in six Amerindian linguistic families: Barbacoa, Choc o, Quechua, Zapara, Aents and Western Tucano. The Barbacoa, whose diversity dates back approximately 3300 years (De la Torre & Balslev, 2008), is comprised of these languages: Awapit, Chafi’ki and Tsafi’ki of Ecuador, as well as Guambiano, Totoro and Barbacoa of Colombia (De Stefano, 1994; Curnow & Liddicoat, 1998). The Tsafi’ki language is still spoken by an indigenous population known as the Tsachilas or Colorados. They live in the Pichincha province, at the Canton of Santo Domingo de los Colorados (Figure S1). Little is known about Tsachilas either ethnologically or historically. Karsten (Karsten, 1988) stated Tsachilas, Cayapas and Cuna of Colombia retain much of the ancient Chibchan culture, which originally spread throughout all what now is Colombia. The languages of these populations show the same basic etymological origins, yet even the separation among them has brought variants in pronunciation (Von Hagen, 1988). The ancient Chibchan culture was subsequently dispersed by tribes coming up the actual Barbacoas, which assimilated the populations. The Tsachilas constitute approximately 2500 people clustered in eight communities. Hunting, fishing and gathering were originally their main activities; however, these activities are no longer practiced, due to a lack of suitable land. Agriculture is still important, but the marketing of agriculture dominates. Currently, they participate in the market economy through livestock, marketing, ecotourism and the practice of traditional medicine. Inbreeding is allowed, and practices within their groups retain community
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traditions, rituals and even their dress. Men manage their hair by bundling it into a cap painted with achiote (Bixa orellana) and oil: this is the reason for the nickname ‘Colorados’. As noted, almost nothing of their traditional territory remains: today, Santo Domingo de los Colorados is an economic hub between the coast and the Sierra, and it represents a collection and marketing centre of a large number of products, so the Tsachilas have been absorbed into the traditional economy of Ecuador (Barfod & Kvist, 1996). This research aimed to analyse the HLA-DQ profile of this population to ascertain its genetic relationships with neighbour groups. Several studies of HLA class I and class II variation in Amerindian populations have been carried out for anthropological as well as evolutionary and clinical studies (Tsuneto et al., 2003; Parolin & Carnese, 2009; Arnaiz-Villena et al., 2010; Vargas-Alarcon et al., 2010; De Angelis et al., 2012). These data were compared with other Amerindian groups and worldwide populations to clarify the relationships among northern inhabitants of South America and to highlight the power of HLA system in determining population relationships.
Materials and method Population sample
This study explores the allele composition of DQA1 and DQB1 genes in the Tsachilas population of the Chiguilpe community. The sample pertains to 52 unrelated healthy individuals; they were blood donors who volunteered for this study after informed consent. Recruiting was carried out in the Chiguilpe area by one of us (GFDS), in accordance with ethical permission standards. DNA was extracted from whole blood using standard protocols (Miller et al., 1998) and was stored at 80°C. Tsachilas samples were compared with other target populations to obtain a pattern of genetic distances useful to test the quality of HLA to ascertain the human relationships and to demonstrate that the analysis of HLA loci is a valuable tool in anthropological studies. The 62 selected populations are shown in Table 1. The choice of populations was guided by the availability of frequency data both for HLA-DQA1 and HLA-DQB1 in the open-source database on www.allelefrequencies.net (Gonzalez-Galarza et al., 2011), that allowed to analyse approximately 11.742 chromosomes. Every population was previously evaluated by anthropological study, and no case–control studies have been included in the analysis to avoid an altered distribution of allelic variants. HLA typing
HLA-DQA1 and HLA-DQB1 loci were high-resolution typed by direct sequencing of exons 2 and 3. The
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Table 1. Reference populations from www.allelefrequencies.net ID
Population
Acronyms
Location
Sample size
References
1 2 3 4 5 6 7 8 9 10 11 12 13
Amhara Bamileke Cameroon Cameroon Saa Congo Kinshasa Bantu Equatorial guinea Bioko Island Bubi Gabon Haut-Ogooue Dienga Gambia Kenya Morocco Oromo Uganda Baganda Zimbabwe Harare Shona
Amh Bam Cam CamS Congo EquaGBB GabonHOD Gambia Ken Mor Oromo Uga Zim
Africa
46 34 126 172 90 100 167 146 144 96 61 47 230
Unpublished Unpublished (Gonzalez-Galarza et al., 2011) (Begovich et al., 2001) (Renquin et al., 2001) (de Pablo et al., 1997) (Migot-Nabias et al., 1999) (Begovich et al., 2001) (Luo, 2006) (Gomez-Casado et al., 2000) Unpublished Begovich et al., 2001; (Louie, 2006)
14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Afroecuadorians Argentina Chiriguanos Argentina Gran Chaco Eastern Toba Argentina Gran Chaco Mataco Wichi Argentina Gran Chaco Western Toba Brazil Central Plateau Xavantes Brazil Guaranı Kaiowa Brazil Guarani M Bya Cayapas Colombia Amazonas Coreguaje Colombia Arhuaco Sierra Nevada Colombia East Amazon Region Nukak Colombia Guajira Peninsula Wayuu Colombia NE plains Sikuani Colombia NW Choco Colombia Providencia Island Africans Colombia S.N. de S.ta Marta Arhuaco Costa Rica and Panama Mexico Sonora Seri Paraguay Ache Quechua Peru Venezuela Sierra de Perija Yucpa
Afroec ArgChi ArgGCET ArgGCMW ArgGCWT BraCPX BraGK BraGMB Cay ColAC ColASN ColEARN ColGPW ColNEPS ColNWC ColPIA ColSMA CosPA MexSS ParA PerQ VenS
America
75 54 135 49 19 74 155 93 74 30 107 20 88 27 20 30 107 56 34 87 44 73
(De Angelis et al., 2012) (Gonzalez-Galarza et al., 2011) (Cerna et al., 1993) (Cerna et al., 1993) (Cerna et al., 1993) (Cerna et al., 1993) (Tsuneto et al., 2003) (Tsuneto et al., 2003) (De Angelis et al., 2012) (Trachtenberg et al., 1996) (Yunis et al., 1994) (Trachtenberg et al., 1996) (Yunis et al., 1994) (Trachtenberg et al., 1996) (Trachtenberg et al., 1996) (Trachtenberg et al., 1996) (Yunis et al., 1994) (Erlich, 2006) (Gorodezky, 2006) (Tsuneto et al., 2003) (Tsuneto et al., 2003) (Layrisse et al., 2001)
36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52
Australia Aborigens Cape York Australia Aborigens Kimberly China Beijing and Xian China Canton Han Cook Island Rarotonga Fiji Viti Levu India Bombay India NE kayastha India Uttar Pradesh Hindu Indonesia Java Yogyakarta region Iran Azeris Iran Baloch Iran Kurds Israel Arab Israel Ashkenazi Israel ethiopian jews Japan
AusACY AusAK ChiBX ChiCH CookRar FijiVL IndB IndNEK IndUPH IndoJYR IranA Iranb IranK IsrA IsraA IsraEJ Jap
Asia
103 41 171 264 78 57 59 190 202 62 100 100 100 109 80 122 50
(Gao, 2000) (Gao, 2000) (Gonzalez-Galarza et al., 2011) (Trachtenberg et al., 2007) (Gao et al., 1992) (Gao et al., 1992) (Begovich et al., 2001) (Agrawal et al., 2008) (Gonzalez-Galarza et al., 2011) (Gao et al., 1992) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Martinez-Laso et al., 1996) (Gonzalez-Galarza et al., 2011) (Geng et al., 1995)
53 54 55 56 57 58 59 60 61 62
Croatia Gorski Kotar Croatia Krk Island Croatia Krk island Dobrinj Czech Gipsy Czech Republic Denmark England Georgia Svaneti Region Svan Greece Crete Spain North Cantabria
Crogk CroKID CroKID CzeG Cze Den Eng Georg GreC SpaNC
Europe
63 212 28 34 106 55 177 80 135 83
(Martinovic et al., 1997) (Gonzalez-Galarza et al., 2011) (Martinovic et al., 1997) (Fernandez-Vina et al., 1992) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Gonzalez-Galarza et al., 2011) (Sanchez-Velasco & Leyva-Cobian, 2001) (Arnaiz-Villena et al., 1999) (Gonzalez-Galarza et al., 2011)
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choice for these regions was due to their crucial role in encoding the HLA molecules; in fact, these exons, respectively, encode for the a1 and b1 regions of the HLA-DQ subunits, which are the regions that form the antigen cleft. The primers for amplification and sequencing are listed in Table S1; they were used according to references (Cordovado et al., 2005; van Dijk et al., 2007; De Angelis et al., 2012). Conditions for the polymerase chain reaction (PCR) assay by custom-designed primers were as follows: 10 min at 95°C, 35 cycles of 30 s at 95°C, 1 min at 53.5°C, 30 s at 72°C and a final extension of 7 min at 72°C. The sequencing reactions used BigDye Terminator v.1.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions and amplification forward primers. All the sequences were compared to the IMGT/HLA Database v.3.5.0 (Robinson et al., 2000) to determine the allelic assignments at the two HLA class II loci. All class II alleles included in the present study are four-digit resolution data. Statistical analysis
Overall distributions of HLA-DQ alleles were determined by the direct counting method. Deviation from the Hardy–Weinberg equilibrium was examined with Arlequin software, version 3.5.1.2 (Excoffier et al., 2005), with the default setting, where the exact P-value is calculated through the Markov chain method (Guo & Thompson, 1992). HLA-DQA1 and HLA-DQB1 haplotypes have been estimated by Phase v. 2.1 (Stephens et al., 2001). To infer relationships between different populations by HLA class II loci, a database of 62 geographically and ethnically different populations has been compiled. Similarity reconstruction with statistical bootstrap involving 1000 replicates was carried out by Nei’s chord distances (Nei, 1987) and neighbour-joining algorithm (NJ), using the PHYLIP v 3.69 software package (Felsenstein, 2009). Genetics distances were also used to draw up a multivariate display of population similarity through multidimensional scaling (MDS). The multivariate analysis reduces multidimensional space to two/three dimensions graphically represented, with minimal loss of information.
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Results and discussion Table 2 shows the HLA-DQA1 and HLA-DQB1 allele frequencies in the Tsachilas population. This points out an expected heterogeneity that encompasses 11 variants at locus HLA-DQA1, with the leading frequency of three alleles, HLA-DQA1*0301 with 15% (n = 16), *0401 with 29% (n = 30) and *0501 with 28% (n = 29), while the other allelic variants are somewhat less represented. The same heterogeneity is shown by locus HLADQB1; in fact, the number of alleles is quite the same, but the frequency distribution is less skewed. The leading presence of HLA-DQB1*0201 (16%) is due to 17 alleles, followed by *0301 with 21 alleles (20%) and the *0302 with 24 alleles (23%) (Table 2). The observed allele frequencies of the HLA-DQA1 and HLA-DQB1 were compared to the theoretically expected frequencies and significant deviations from the Hardy–Weinberg equilibrium and were observed especially for the HLA-DQA1 locus (DQA1: P = 0.00000; DQB1: P = 0.00005). It is interesting to note the high frequency of HLADQA1*0401 in the sample that might be evidence of putative genetic protection against helminth infections, in particular onchocerciasis, although no individual data are available about the presence/absence of disease (De Angelis et al., 2012). The establishment of a new, increasing focus on onchocerciasis was suspected in the Tsachilas because of the immigration of Cayapas Amerindians from hyperendemic onchocerciasis focus over the last 20 years (Cooper et al., 2001) and the presence of the vector species Simulium exiguum (Charalambous et al., 1997). The random sampling of apparently healthy people might be inclusive of people protected against onchocerciasis carrying the protective allele DQA1*0401. No haplotype is markedly frequent in the sample, as reported in Table S2. These findings highlight the absence of an evident linkage disequilibrium pattern in the Amerindian sample. To test the Tsachilas relationships with other human groups, the distribution of HLA-DQ alleles was compared to other populations by MDS and NJ dendrogram. The population choice was arbitrarily carried out on the basis of geographical distribution and
Table 2. DQA1 and DQB1 allele frequencies in Tsachilas DQA1
*0102
*0201
*0301
*0302
*0303
*0401
*0402
*0501
*0502
*0503
*0601
TOT
N alleles % S.E.
4 3.8 0.019
5 4.8 0.021
16 15.4 0.035
3 2.9 0.016
2 1.9 0.013
30 28.8 0.044
5 4.8 0.021
29 27.9 0.044
3 2.9 0.016
1 1 0.009
6 5.8 0.023
104 100
DQB1
*0201
*0202
*0301
*0302
*0303
*0304
*0401
*0402
*0501
*0502
TOT
N alleles % S.E.
17 16.3 0.036
6 5.8 0.023
21 20.2 0.039
24 23.1 0.041
9 8.7 0.028
2 1.9 0.013
8 7.7 0.026
2 1.9 0.013
8 7.7 0.026
7 6.7 0.024
104 100
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depending on the availability of comparable data for both analysed loci. The analyses were separately carried on for each locus (HLA-DQA1 and HLA-DQB1) and then used by the pooled loci (HLADQA1 + HLA-DQB1). Despite some inconsistencies probably attributable to the limited number of markers, the MDS made on data from the DQA1 locus reveals the presence of four main clusters: roughly due to ethnogeographic groups of Asians, South Americans, Europeans and Africans (Figure S2). Indeed, the Amerindians are well clustered at the edge of the distribution by Dimension 1, with variable eigenvalues by Dimension 2 and Dimension 3. Tsachilas are placed in the middle, suggesting a genetic contribution by both Central Americans (Costa Rica and Panama; Mexico) and northern South American human groups (Colombian Amerindians; Brazilians). The clustering of the other ethnogeographic groups is less defined than Amerindians. Further discussion can be made about the similarity between the African peoples and Afroecuadorians, presumably due to the genetic origin of the African communities of Ecuador (De Stefano, 1994; MartinezLabarga et al., 1999), as the result of the settlement of human groups conducted in the Americas by the slave trades from Guinea Gulf in the seventeenth century (De Stefano, 1994). The MDS for the HLA-DQB1 (Figure S3) locus shows that the ethnogeographical differences are less defined, probably due to selective convergence; the clear scenario has been replaced by a more nuanced situation, and the Tsachilas sample is located at the edge of American group. This could be due to functional constraints of the peptide encoded by HLA-DQB1: the section of the HLA-DQ heterodimer essential to the housing of the antigenic peptide within the binding cleft of the molecule (Menconi et al., 2010). Thus, it could be estimated how this locus might be subjected to a wider selective force, resulting in a loss of quality in the discriminatory inference of population relationships. The NJ dendrograms made by standard Nei genetic distances on both loci and 1.000.000 permutations are consistent with MDS representations. The MDS of both the loci highlights, once again, the presence of four different clusters, with better resolution; the improvement is a consequence of both loci having been considered (Figure 1). The graph reveals the remarkable diversity of the American population, which is made up of different ethnic groups (De Stefano, 1994; Polimanti et al., 2011). This is also highlighted in Figure 2, in which only new world groups are compared. The MDS plot highlights (stress = 0.12) a clear differentiation among populations that might be related to the origin of each human group. Tsachilas lie in the right upper quadrant, along with Central Amerindians and northern South Amerindians, and this relatedness may be a detector of their linguistic similarity.
Figure 1. Tridimensional multidimensional scaling (MDS) HLADQA1+HLA-DQB1; Stress = 0.14. Black dots are Africans, white dots are South Americans, black squares are Asians and white squares are Europeans. Tsachilas sample is marked by a black star.
First dimension shows a cluster for Argentinians and Brazilians that refers to people living around Chaco region, that is a central South American area boundaried by Southern Brazil, Northern Argentina, Bolivia and Paraguay. Nowadays, this territory is largely uninhabited except for different hunter-gatherer peoples, making the area a great cultural gathering and genetic exchange (Cabana et al., 2006). Colombians are more scattered in the plot because of their peculiar genetic background. This scenario has already been suggested by mitochondrial DNA studies showing asymmetrical Amerindian admixture in Colombia. Amerindians should experience weak matrilineal admixture with Caucasians or Africans that reached the area, resulting in different patterns of past ethnic admixture in the geographic region now encompassing Colombia, which is also reflected in much of the region’s cultural diversity (Rodas et al., 2003). The tight genetic proximity between Tsachilas and Costa Rica/Panama sample is worthy of consideration: these areas are considered the centre of Chibchan languages (Greenberg, 1987). The reported Central Amerindians are linguistically clustered in the Macro-Chibchan family (Rhulen, 1991), while the Tsachilas are a Barbacoan-derived people from the ancient Yumbos human group (Lippi & Gudi~ no, 2004). According to archaeological research, the Yumbos occupied southwestern Colombia and northeastern Ecuador. Less than 10% linguistic correspondence between Macro-Chibchan and Barbacoa languages was detected, and doubts on the rightness of the Macro-Chibchan group have been advanced (Wheeler, 1972; Constenla, 1991). It could be hypothesized that Barbacoan might be a mix of Chibchan language and languages from the Andes. On the basis of this hypothesis, it has been proposed that the Chibchan populations moved southwards and mixed with
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Figure 2. Bidimensional multidimensional scaling (MDS) HLA-DQA1+HLA-DQB1 of Central Amerindians samples; Stress = 0.12.
Andeans (Lippi, 2003). The genetic affinity between Tsachilas and Costa Rican sample might account this hypothesis: Tsachilas could represent a Chibchan group southwards moving with weaker genetic admixing than other Chibchan-derived people as the Cayapas Amerindians are (Von Hagen, 1988). Archaeological data confirm that Barbacoans split from the Macro-Chibchan southward from Central Colombia, and they reached south Colombia, northern Ecuador and the western slope of the Andes. Therefore, Barbacoans represent southern populations closer to Chibchan, and they probably mixed with Andean human groups. Moreover, according to these data, Lippi proposed the ancient Yumbos derived from the Caras, that is, an Ecuadorian highplane, prehispanic population (Lippi, 2003). Some Spanish reports identified South and North Yumbos: North Yumbos would seem to have derived from Caras and afterwards would have reached Western Pichincha, according to ‘Toras’ (archaeological buildings). The above results may support the hypothesis suggested by some authors (Melton et al., 2007), concerning an expansion of the Chibchan-speaker groups from the central to southern portion of the continent. Subsequently, the phenomena of genetic drift would produce the considerable genetic diversity that is observed in allele frequencies among different populations. While archaeological and linguistic research has found a cultural association between Central and South American Chibchan-speaking populations, the biological relationship has long remained largely unresolved. This study demonstrated that northern South and lower Central American Chibchan speakers might share a similar genetic structure, but if they share a common ancestry it occurred in the distant past.
Conclusions Several polymorphic regions of the human genome have been used in recent years to determine human
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population relationships and, like all the preferred regions that exhibit a high degree of polymorphism, the HLA loci could be used for this aim. Overall, the analysis of the two HLA class II loci has depicted a geo-ethnic distribution indicating that the presence of numerous alleles (against biallelic SNPs, mtDNA and Y chromosome) does not interfere with the phylogenetic information content of the loci, provided that the frequency distribution of the populations is significantly different. Instead, it increases the chance of the presence of signature alleles and specific haplotypes in cases of closely linked loci, like HLA. The present study has allowed us to demonstrate the effective discriminatory potential of the two HLA loci; in fact, the results show that the clusters might reflect the origin and the geographical distribution of the populations, highlighting also genetic relationships that could be modified by historical and cultural events such as migration or admixture. Indeed, HLA analysis might be an aid in population analysis, achieving similar results than mtDNA. However, there are potential problems associated with the use of HLA loci in inferring human population relationships. Most important is the nonuniformity of HLA typing methodologies, as some studies are based on low-resolution typing while others are based on high-resolution typing. Preferably, if all the HLA loci (both class I and class II) are analysed with a high resolution of all the alleles and a proper statistical interpretation based on more logistic approaches is carried out, then the HLA loci can be an ideal marker to infer genetic differences between inter- and intra-geo-ethnic groups. Moreover, if the property of rarity of the recombination at the HLA loci is fully explored to assign extended haplotypes, then there will be a high possibility that the HLA loci could reconstruct the human phylogenies as exactly and as accurately as those deciphered by the mtDNA and Y chromosome markers. This attempt may be useful to develop a new aspect of the HLA studies,
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although the disease association ones are, to date, the leading fieldworks in HLA genetics.
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Supporting Information Additional supporting information may be found in the online version of this article: Table S1 PCR and sequencing primers. Table S2 Haplotype HLA-DQA1 and HLA-DQB1 in Tsachilas. Figure S1 Geographical location of Canton de Santo Domingo de los Colorados.
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Figure S2 Tridimensional MDS for HLA-DQA1, based on Nei’s chord distances; Stress = 0.13. Black dots are Africans, white dots are South Americans, black squares are Asians and white squares are Europeans. Tsachilas sample is marked by a black star.
Figure S3 Tridimensional MDS HLA-DQB1 based on Nei’s chord distances; Stress = 0.12. Black dots are Africans, white dots are South Americans, black squares are Asians and white squares are Europeans. Tsachilas sample is marked by a black star.
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