AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 125:85– 89 (2004) DYS19 and DYS199 Loci in a Chilean Population of Mixed Ancestry L. Cifuentes,1* R. Morales,2 D. Sepúlveda,3 H. Jorquera,2 and M. Acuña1 1 Programa de Genética Humana, ICBM, Facultad de Medicina, Universidad de Chile, Santiago 7, Chile Laboratorio de Biologı́a Molecular, Departamento de Laboratorios, Servicio Médico Legal, Ministerio de Justicia, Santiago, Chile 3 Genética y Tecnologı́a Ltda., Santiago, Chile 2 KEY WORDS DYS19; Y microsatellite; DYS199; Y-chromosome haplotype; Chilean population; genetic admixture; asymmetric mating system ABSTRACT The current Chilean population originated from admixture between aboriginal populations (Amerindians) and Spanish conquerors of European origin. Consequently, the unions that gave rise to the Chilean population were chieﬂy between Spanish males and aboriginal females, and not the converse. To test the hypothesis that the Y chromosome of the Chilean population is mainly of Spanish origin, while the other chromosomes are from mixed (European and aboriginal) origin, we studied the DYS19 and DYS199 loci in two samples. One sample was obtained from a high socioeconomic stratum, while a second sample was from a low stratum. We studied male blood donors (N ⫽ 187) from Santiago, the capital of the country. Subjects were typed for the autosomal ABO and Rh (locus D) blood groups, and for the Y-linked DYS19 and the DYS199 loci, reported as Y-chromosome haplotypes. The aboriginal admixture was estimated for each genetic marker. The percentage of aboriginal admixture The Chilean population was generated by the admixture between aboriginal populations (Amerindians) and Spanish conquerors of European origin who arrived in the country in the latter part of the sixteenth century (Encina, 1983). The Spanish conquerors who arrived in the Chilean territory in the early years of the colonization (roughly from the late 1500s to late 1600s) were mainly males. Some Spanish females arrived later (at the end of the sixteenth century), but they only accounted for a small fraction of the Spanish population (Thayer-Ojeda, 1919). Adult male mortality was probably higher during the period of conquest than adult female mortality due to a variety of factors, including warfare with Europeans. This demographic background predicts that the unions that gave rise to the Chilean population were chieﬂy between Spanish males and aboriginal females, and not the converse. While a few Spanish females accompanied the males arriving in South America, unions between aboriginal males and Spanish females would have been rare. Fairly small groups of other immigrants settled later in the country. For example, Croatians settled in the © 2004 WILEY-LISS, INC. was 38.17% for the ABO system and 31.28% for the Rh system in the low socioeconomic stratum and 19.22% and 22.5%, respectively, in the high stratum. Y-chromosome haplotype frequencies constructed from the DYS19 and DYS199 loci demonstrated that the main haplotypes were DYS19*14/DYS199 C, as is often the case with many European populations, and DYS19*13/DYS199 C. The aboriginal admixture from Y-haplotype frequencies was estimated to be 15.83% in the low socioeconomic stratum and 6.91% in the high stratum. These values are lower than the values found using autosomal genetic markers, and are consistent with the historical background of the population studied. This study highlights the population genetic consequences of the asymmetric pattern of genome admixture between two ancestral populations (European and Amerindian). Am J Phys Anthropol 125:85– 89, 2004. © 2004 Wiley-Liss, Inc. southernmost part of Chile in the beginning of the twentieth century, and Germans arrived around the middle of the nineteenth century in another region of the South. Although these groups were more balanced in terms of male-female ratio, their genetic contribution was not great enough to alter the asymmetry that characterized the original admixture pattern. Thus it follows that the Y chromosomes of the Chilean population must be mainly of Spanish origin, while autosomal chromosomes must be of Grant sposnor: D.I.D., Universidad de Chile; Grant number: ENL2000/03. *Correspondence to: L. Cifuentes, Programa de Genética Humana, ICBM, Facultad de Medicina, Universidad de Chile, P.O. Box 70061, Santiago 7, Chile. E-mail: firstname.lastname@example.org Received 29 August 2001; accepted 21 July 2003. DOI 10.1002/ajpa.10380 Published online 12 January 2004 in Wiley InterScience (www. interscience.wiley.com). 86 L. CIFUENTES ET AL. mixed (European and aboriginal) origin (Cifuentes et al., 1999). Many population studies have been conducted on the Chilean population of mixed origin; all were based on genetic markers located on autosomal chromosomes (Cifuentes et al., 1988). They showed that the present Chilean population has an aboriginal (Amerindian) admixture of around 40% (Valenzuela et al., 1987). This percentage depends on the socioeconomic stratum: it is fairly small (typically less than 10%) in the upper strata, and greater in lower strata (Valenzuela, 1988). Genetic markers located in nonautosomal regions of the genome have not been studied in the Chilean mixed population. The DYS19 locus is a microsatellite located in the short arm of the Y chromosome. Its population diversity is due to a variable number of GATA tandem repeats (Santos et al., 1993), from 186 bp (13 or A allele) to 202 bp (17 or E allele) (Roewer et al., 1992); there are also other minor alleles (Z). The 13 or A allele from this locus is the most common allele in Amerindian populations, while the 14 or B allele is the most common in European populations. The DYS199 locus is a single-base change (C 3 T). The T allele has been found only in Native American populations, where it is predominant and is in linkage disequilibrium with the 186-bp allele of the DYS19 locus (Underhill et al., 1996). The goal of this paper is to study the DYS19 and DYS199 loci in two Chilean population samples of mixed ancestry, and compare the magnitude of the aboriginal admixture based on these two genetic markers, which are located in the nonrecombinant region of the Y chromosome, with estimates based on autosomal genetic markers. We chose these genetic markers because their allele frequencies are quite different in the Spanish (González-Neira et al., 2000) and South American aboriginal populations (Santos et al., 1996; Bianchi et al., 1997; Underhill et al., 1996), which make them a reliable tool to estimate the ethnic admixture in the Chilean population. SUBJECTS AND METHODS Two samples of male blood donors from Santiago, the capital of Chile, were studied. One sample consisted of male blood donors from a low socioeconomic stratum (according to a standard questionnaire; Sepúlveda, 1960) who visited the San José Hospital in the northern area of Santiago (N ⫽ 99) (Acuña et al., 2000). The second sample consisted of male blood donors from a high socioeconomic stratum who visited the Las Condes Clinic (N ⫽ 88). An appropriate informed consent was obtained from each donor, following the guidelines of the University of Chile School of Medicine Ethics Board. They were typed for the ABO and Rh (locus D) blood groups according to standard procedures. DNA was extracted from blood samples using the organic method described by Comey et al. (1994). The DNA was quantiﬁed by means of electrophoresis, employing 1% agarose gels, ethidium bromide staining, and comparison with known standards of molecular weight. To genotype the DYS19 locus, ampliﬁcation was made from a 50-ng DNA template, using the primers described by Roewer and Epplen (1992). As a negative control, we simultaneously ampliﬁed a DNA sample of a female. The polymerase chain reaction (PCR) was carried out in a Perkin-Elmer thermal cycler for 25 cycles. Each cycle consisted of 30 sec at 94°C for denaturation, 30 sec at 65°C for primer annealing, and 90 sec at 72°C for primer extension. After ampliﬁcation, the samples were stored at 4°C. Samples were resolved in 6% native polyacrylamide gels. Following electrophoresis, fragments were visualized using highly sensitive silver staining, according to the method of Budowle et al. (1995). Allele identiﬁcation was carried out by comparing each fragment against an allelic ladder constructed from a mixture of the different alleles detected in several individuals. The DYS199 locus was typed by means of two ampliﬁcation reactions for each sample, using between 15–25 ng of DNA with the speciﬁc primers described by Underhill et al. (1996). Thirty-ﬁve cycles using the following twostep PCR regime were performed: 94°C for 30 sec, and 61°C for 20 sec. Ten microliters of the speciﬁc 201-bp product were run in an 1% agarose gel, and UV-visualized by ethidium bromide-staining. Gene frequencies for the ABO and Rh system were estimated according to the Bernstein method, assuming Hardy-Weinberg equilibrium (CavalliSforza and Bodmer, 1971). The frequencies for Ychromosome haplotypes constructed from the DYS19 and DYS199 loci were estimated by direct count (Nei, 1987), and the standard errors were calculated assuming a binomial distribution (Steel and Torrie, 1980). The percentage of aboriginal (Amerindian) admixture in each genetic marker was estimated using Bernstein’s method (Cavalli-Sforza and Bodmer, 1971), based on gene frequency data from ancestral populations already published. These estimates were made based on the assumption that allele frequencies from current Spanish and Amerindian populations are similar to those of the Spanish conquerors and Amerindians who met in the Chilean territory ﬁve centuries ago. RESULTS Table 1 shows the frequencies for the Y-chromosome haplotypes constructed from the DYS19 and DYS199 loci in the Chilean samples (San José Hospital, HSJ; Las Condes Clinic, CLC). Table 1 demonstrates that the main haplotype was DYS19*14/ DYS199 C (as is often the case with European populations). Nevertheless, the presence of the DYS19*13/DYS199 T haplotype in these two Chilean mixed samples is an evidence of an Amerindian ethnic component, mainly in the HSJ sample. The T allele from the DYS199 locus (which has been reported only in Amerindians populations) had a very 87 DYS19 AND DYS199 LOCI IN CHILEAN POPULATION TABLE 1. Y-chromosome haplotype frequencies constructed from DYS19 and DYS199 loci in two Chilean samples of mixed ancestry San José Hospital (N ⫽ 99) Haplotype DYS19*12/DYS199 DYS19*13/DYS199 DYS19*14/DYS199 DYS19*15/DYS199 DYS19*16/DYS199 DYS19*17/DYS199 DYS19*13/DYS199 DYS19*14/DYS199 DYS19*15/DYS199 DYS19*16/DYS199 DYS19*17/DYS199 C C C C C C T T T T T Las Condes Clinic (N ⫽ 88) Frequency Standard error Frequency Standard error 0.0110 0.2198 0.5275 0.1429 0.0550 0.0000 0.0219 0.0000 0.0219 0.0000 0.0000 0.01093 0.04341 0.05234 0.03668 0.02389 0.00000 0.01537 0.00000 0.01537 0.00000 0.00000 0.0000 0.0455 0.6705 0.1363 0.1023 0.0000 0.0227 0.0227 0.0000 0.0000 0.0000 0.00000 0.02220 0.05011 0.03658 0.03230 0.00000 0.01589 0.01589 0.00000 0.00000 0.00000 TABLE 2. Gene frequencies of ABO and Rh system and percentage of aboriginal admixture in two Chilean samples of mixed ancestry1 Gene frequencies in San José Hospital Allele Spanish2 Aboriginal3 Gene frequency A B O Rh D Rh d 0.2864 0.0670 0.6465 0.5937 0.4063 0.0678 0.0000 0.9322 1.0000 0.0000 0.2143 0.0354 0.7575 0.7202 0.2798 Las Condes Clinic Aboriginal admixture (%) Gene frequency Aboriginal admixture (%) 32.98 47.16 38.85 31.13 31.13 0.2395 0.0455 0.7149 0.6855 0.3145 21.45 32.09 23.94 22.59 22.59 1 San José Hospital, low socioeconomic stratum; Las Condes Clinic, high socioeconomic stratum. Campillo (1976). 3 Llop and Rothhammer (1988). 2 low frequency in both Chilean samples. The high frequency of the DYS19*13/DYS199 C haplotype observed in HSJ is surprising: it is considerably greater than that of either of the supposedly ancestral populations. Table 2 shows the gene frequencies of the Rh and ABO blood groups for the two samples studied and the estimates of aboriginal admixture in the samples studied, based on these two genetic systems. These autosomal genetic markers are useful tools to estimate the genetic admixture between European and Amerindian populations because of the large differences in allele frequencies between these two ethnic groups. The gene frequencies from the Spanish and Amerindian populations used to estimate the aboriginal admixture are those published by Campillo (1976) and Llop and Rothhammer (1988). The percentage of aboriginal admixture was 38.17% for the ABO system (doing a weighted average using the admixture estimate of each allele) and 31.13% for the Rh system in HSJ (low socioeconomic stratum), and 23.71% and 22.59% in the CLC sample, respectively. These values are consistent with the admixture estimates published for different socioeconomic strata in the Chilean population (Valenzuela, 1988). Table 3 shows the percentage of aboriginal admixture in the samples based on haplotype frequencies from the DYS19 and DYS199 loci. To estimate the aboriginal admixture, we collected data from the literature for each ancestral population: the South American aborigines (Underhill et al., 1996; Bianchi et al., 1997, 1998; Di Pierri et al., 1998) and the Spanish population (Gonzalez-Neira et al., 2000; Kayser et al., 1997). The weighted average percentages of aboriginal admixture in the HSJ sample were 15.83%, and 6.91% in the CLC sample. It can be seen that the aboriginal admixture is lower in the high socioeconomic stratum sample (CLC), in accordance with data already published for autosomal loci (Valenzuela, 1988). The percentage of aboriginal admixture is lower in the Y-chromosome loci than the percentages found in the autosomal loci for both samples studied. DISCUSSION The Y-chromosome haplotype frequencies based on the DYS19 and DYS199 loci in the Chilean samples are consistent with the historical background of the population under consideration. The high frequency of the DYS19*14/DYS199 C haplotype is evidence of a preponderant European (Spanish) component. However, the DYS19*13/DYS199T haplotype reﬂects the aboriginal (Amerindian) components. We cannot explain the high frequency of the DYS19 *13/DYS199 C haplotype observed in HSJ; this frequency is greater than those reported for European populations. This result cannot be explained by technical errors, for if that were the case we should also have observed the same result in the sample from Clı́nica Las Condes. While it is possible that the original inhabitants of this region of Chile had allele frequencies different from those of the South American Indians used as the reference pop- 88 L. CIFUENTES ET AL. TABLE 3. Percentage of aboriginal admixture in Y-chromosome haplotypes constructed from DYS199 and DYS19 loci of a Chilean mixed population, based on comparison with allele frequencies in Spanish and Amerindian populations1 Frequencies in populations Chilean mixed samples Haplotype DYS19*12/DYS199 DYS19*13/DYS199 DYS19*14/DYS199 DYS19*15/DYS199 DYS19*16/DYS199 DYS19*17/DYS199 DYS19*12/DYS199 DYS19*13/DYS199 DYS19*14/DYS199 DYS19*15/DYS199 Total2 C C C C C C T T T T Aboriginal admixture(%) Spanish Amerindian HSJ CLC HSJ CLC 0.0000 0.0942 0.6993 0.1449 0.0435 0.0181 0.0000 0.0000 0.0000 0.0000 276.0 0.0000 0.0101 0.0202 0.0202 0.0000 0.0000 0.0101 0.7980 0.1212 0.0202 99.0 0.0110 0.2198 0.5275 0.1429 0.0550 0.0000 0.0000 0.0219 0.0000 0.0219 99.0 0.0000 0.0455 0.6705 0.1363 0.1023 0.0000 0.0000 0.0227 0.0227 0.0000 88.0 0.00 ⬍0.00 25.30 1.60 ⬍0.00 100.00 0.00 2.70 0.00 ⬎100.00 99.0 0.00 57.91 4.24 6.90 ⬍0.00 100.00 0.00 2.85 18.73 0.00 88.0 1 HSJ, San José Hospital; CLC, Las Condes Clinic. Number of Y chromosomes from each population. Frequencies for Spanish and Amerindian populations correspond to pooled data already published (see text). 2 ulation, or that there has been some selective force operating to favor this haplotype, there is currently no available evidence to support either of these hypotheses. Another possibility is the postcolonial incorporation of an atypical European-descent population to the northern area of Santiago. However, there is no historical information to support this idea. The percentage of aboriginal admixture found in the Chilean samples for Y-chromosome haplotypes based on the DYS19 and DYS199 loci is much lower than the percentage of Amerindian ethnic component described for autosomal regions of the genome in the Chilean population. Again, this result agrees with the historical background of the population studied, which indicates an asymmetrical system of mating between ancestral populations. This asymmetry also predicts that the proportion of the indigenous component in the mixed Chilean population should be greater for genes located in the X chromosome, and greater still for mitochondrial genes. This hypothesis is supported by a recent study of polymorphisms in mitochondrial DNA (Rocco et al., 2002), which reported that 84% of the haplogroups in a similar mixed Chilean population were of Amerindian origin. This situation may also be found in other populations of mixed origin. Previous studies in other South American populations with an aboriginal component greater than that found in the Chilean population also demonstrated directionality of marriages. For example, the genetic admixture with Amerindians is much lower for the Y chromosome than for other regions of the genome in the Peruvian Quechuas (Rodrı́guez-Delﬁn et al., 2001). A similar situation was reported in terms of the contribution of indigenous females to the formation of the Belém population (Brazilian Amazon region). Here, it was found that the contribution of females is 10 times higher than that of indigenous men (Dos Santos et al., 1999). This study demonstrated one of the consequences, in terms of population genetics, of an asymmetric pattern of genome admixture between the ancestral European and Amerindian source populations. The autosomal Amerindian component of the Chilean population is around 40%, while the Amerindian Y component appears to be less than 20%. In the case of the Chilean population, we suggest that it is important to consider the different patterns of ancestry of the different regions of the genome when making risk assessment of Y chromosome-based genetic diseases that depend on ethnic origin (Osterlund et al., 2000). 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