The STR polymorphism ( AAAAT ) n within the intron 1 of the tumor protein 53 ( TP 53 ) locus in 17 populations of different ethnic groups of Africa , America , Asia and Europe

The STR (AAAAT)n within intron 1 of the TP53 locus was screened in 17 populations from 3 main ethnic groups: Europeans, Asiatics, and Africans, and from the hybrid population of Costa Rica (1968 samples). Three alleles, 126/7 (bp/copies of the repeat), 131/8 and 136/9 were the most prevalent in all populations. Other alleles rarely reached frequencies of 10% or higher. Observed heterozygosities ranged between 0.351 and 0.829. Patterns of diversity fit well with both the geographic origin of the samples and the history of the populations screened. A statistical test suggests that single-step mutational events have been the main mechanism producing new alleles at this locus. Fixation indexes (RST) for this marker showed an effect of population subdivision on divergence only within the Asiatic group; they were insensitive at the level of major ethnic groups as well as within Africans and within Europeans. Rev. Biol. Trop. 52(3): 645-657. Epub 2004 Dic 15.

The tumor protein 53 locus (TP53) (MIM No.191170, GOB ID 120445) maps to 17p13.1 (Isobe et al. 1986).The gene spans 12.5 kb, is segmented in I I exons, the first of which is not translated, and codes for a 43.5 kDa protein (p53) involved in cellular responses to genetic damage as mediator in DNA repair, in growth arrest at the GI-S checkpoint of the cell cycle, or as an inductor of apoptosis (Hesketh 1997, Janus et al. 1999).Although several polymorphic regions have been described within the locus, population genetic studies are not abundant.Exceptional are the works of two groups which have studied the haplotypic variation defined by the presence/absence of a 16bp duplication within intron 3, of a BstU I restriction site within axon 4 and of an MspI restriction site within intron 6 in Northern Europeans, Chinese and Africans (Sjalander et a/. 1995(Sjalander et a/. , 1996)), and in Amerindians, Blacks and European descendants from Brazil (Gaspar et al. 2001(Gaspar et al. , 2002)).On the other hand, several mutations have been directly implicated in approximately 50% of all human cancers (Janus et al. 1999) -for an extensive review see OMIM.In this association, Ridanpaa et al.
REVISTA DE 1310LOGIA TROPICAL (1995) have studied samples of archival lung cancer tissue using a pentanucleotide (AAAAT)n within the 6.1 kb-long intron 1 of the locus, a polymorphism first reported by Entreat et al. (i 991) and further characterized by Hahn et al. (1995).The methodological improvements developed by Hahn et al. (1993) facilitated the screening of large samples.Hence, population data of this polymorphism have been reported for two samples, one from Germany (Hahn et al. 1995) and the other from Northern Portugal (Gusmio et al. 1997).
In this study, new population data of this STR are reported for 17 populations of 3 major ethnic groups, Europeans, Asiatics and Africans.The data permitted documentation of some of the effects caused by the human spread through the world on the diversity patterns of this STR, and also to statistically explore the main mutational mechanism from which new alleles originate and to test the potential of this marker in phylogenetic studies.

MATERIALS AND METHODS
Data were independently obtained by two groups, one from the Section of Human Genetics, Institute for Health Research (INISA), University of Costa Rica, Costa Rica, and the other from the Institute of Biochemistry, Justus-Liebig University at Giessen, Germany.We decided to pool our results to achieve a wider perspective on the distribution and evolution of the polymorphism; therefore, some methodological differences in laboratory methods are to be noted.

Populations
Samples analyzed in Costa Rica are from 10 Amerindian populations, 1 group of Afro-Costa Ricans and 1 sample of the general Costa Rican population.Groups screened in Germany are 2 from Africa, 1 from Germany, 1 from Turks living or born in Germany and I from The Philippines.The populations were grouped into 3 major ethnic categories: (1) Asiatics, the Amerindians and the Filipinos; (2) sub-Saharan Africans (in this paper rcfered as Africans), the Namibian, Ugandan and the Afro-Costa Ricans; (3) Europeans, the Germans and the Turks.Data for the same marker from Northern Portugal reported by Gusmao et al. (1997) were added to the European group.The general Costa Rican population was considered as hybrid because of its multiethnic origin (Morera et al. 2001(Morera et al. , 2003)).1991).An additional sample of 75 Amerindians was obtained in Matambu, Costa Rica, from a group whose ancestors belonged to an area of Mesoamerican (Central Amerind) cultural tradition and their now extinct language belonged to the Oto-Manguean Family (e.g.Newson 1987).Sampling methods were as described elsewhere (Barrantes et al. 1990)

DNA extraction
DNAs analyzed in Costa Rica were extracted from whole peripheral blood (ACD) either with phenol-chloroform (Hermann and Frischauf 1987) or with NaCI (Miller et al. 1988).DNAs screened in Germany were also extracted from whole blood (EDTA) with phenol-chloroform.

PCR conditions
a. Conditions employed in Costa Rica.The oligonucleotide primers used were those described by Hahn et al. (1993).PCR-conditions were those of Ridanpaii et al. (1995) but scaling down reaction volumes to 250 instead of 50 µI.
b. Conditions employed in Germany: Two combinations of primers were used after Hahn et al. (1993Hahn et al. ( , 2001)).PCR conditions were according to Hahn et al. (2001).

Separation of PCR products
a. Conditions employed in Costa Rica.Native PAGE (3% stacking, 2cm below the REV1STA DE I3IOLOGIA TROPICAL comb level, and 10% resolving).Gels were stained with silver nitrate.
b. Conditions employed in Germany.Native and denaturing PAGE, and, denaturing capillary electrophoreses (Hahn et al. 2001).

Statistical analyses
Allele frequencies were estimated by gene counting and their standard deviations were calculated assuming a multinomial distribution; gene diversities were calculated after Nei (1987).The null hypothesis, that mutation occurs at one step events, was tested using the Markov-chains recursive method (Griffiths andTavare 1994, Nielsen 1997) which takes the form of a likelihood ratio; i.e. the probability of a one-step model relative to that of a multi-step model (-2log[L(0,q 41)/L(0,q)1), where 0 = 4Neit, Ne is the effective population size, t the mutation rate (Ohta and Kimura 1973) and q the proportion of multi-step mutations (0 at one-step).Nielsen (1997) showed that if q = 0 the likelihood ratio resembles a x2 distribution when sample sizes are large enough.0 was estimated by maximum likelihood and both components of the likelihood ratio were calculated by series alone million iterations using the Microsatellite Analysis (M1SAT) Program (Nielsen 1997).

RESULTS
The absolute and relative phenotype frequencies obtained in the Asiatic group are presented in Table 1.Those found in the Africans, Europeans and the Hybrid population of Costa Rica are shown in Table 2. Their corresponding allele frequencies, observed and expected (gene diversities) heterozygosities and their Hardy-Weinberg fit are summarized in Tables 3 and 4.
Although three alleles (126, 131, 136 bp) are the most frequent in all populations, the number of phenotypes and gene diversities follow patterns concordant with the geographic origin of each population as if they had spread from an African centroid.Africans, the oldest of them under this perspective, have, as would be expected, more alleles (7-9) more phenotypes (18-19) and higher gene diversities (0.739-0.784) than the other groups.Europeans, have 4-7 alleles, 6-14 phenotypes and gene diversities ranging from 0.55-0.705.Amerindians have 3-6 alleles, the number of phenotypes ranged between 3 and 12 and gene diversities between 0.377 and 0.729.However, setting apart the Guatuso, Huetar and Chorotega, tribes with the highest degree of interethnic admixture (Barrantes 1993), the rest of the tribes show an even lower diversity: 3 alleles, the number of phenotypes is 4 or 5 and the gene diversities range between 0.351 and 0.697.Filipinos have 9 alleles, 16 phenotypes and a gene diversity of 0.704, values intermediate between Africans and Europeans.The hybrid population of Costa Rica reflects, through its high diversity, 7 alleles, 20 phenotypes and a gene diversity of 0.705, its multiethnic origin.
Genetic distances (Table 5) and a multidimensional analysis (Fig. 1) were performed to obtain a complementary perspective of the path followed by this STR during the divergence of these populations and to test the confidence of the data obtained from the samples analyzed (however, these statistics were not intended to estimate the actual genetic distances among the populations compared, i.e. they must be regarded in their qualitative sense).Populations clustered in three groups, an array in agreement with expectations according to their geographic origin and  history: the Europeans, the Amerindians and a third one including the Africans, the Filipino and the hybrid population of Costa Rica.The fixation indexes for the major groups, Europeans, Asiatics, Africans and hybrid were Ris = 0.0112 (p < 0.001), R1 =, 0.0197 (p < 0.001) and RsT = 0.0085 (p > 0.05).Thus variation is significant among individuals within subpopulations (R15) as well as among individuals within the total of all populations (RIT).In contrast, R$T did not disclose any significant effect of population subdivision on the  divergence of this STR at this level of population subdivision.The same was the case within Africans (RST = 0.0015, p > 0.05) and within Europeans (RST = -0.0005,p > 0.05).However, within the Asiatics RST showed that subdivision in the three possible combinations has been significant: within the Chibchan (RsT = 0.1411, p < 0.001), Chibchan total vs. Mesoamerican (RsT = 0.0199, p < 0.05), and Chibchan total vs. Mesoamerican vs. Filipino (Rsi.= 0.0324, p< 0.001).
The data obtained also permitted a statistical exploration of the possible molecular mechanisms originating new alleles at this REVISTA DE BIOLOG IA TROl'ICAL STR.Expansion or contraction in the number of tandem repeats could occur by single-or by multi-step events, i.c. the original allele could win or lose a single repeat or multiple repeats per mutational event.Table 6 summarizes likelihood ratios for a one-step mutation model relative to a multi-step mutation model in ten populations (the Chibchan tribes were pooled as a single group).All ratios indicate that the single-step model better explains the observed allele-frequency distributions.Therefore, as 0 measures the joint effect of mutation, through its rate, and population structure, through Ne, it would be safe to postulate that mutation on this marker acts mainly by single steps.

DISCUSSION
The characterization of genetic variation at an STR, the (AAAAT).within intron I of the TP53 locus in 17 populations of 3 major ethnic groups, permitted the documentation of some of the effects of the spread of the human species through the world on its patterns of diversity.The subdivisions of ancestral populations, mainly by fissions, were events often associated with bottlenecks.Since-the derived groups were small groups of related individuals, one of the main consequences during differentiation was the loss of alleles by genetic drift (Fix 1999).In the groups screened here the reduction of diversity follows an almost linear fashion from the African Continent: Africans, the oldest and most diverse, Europeans and Filipinos intermediate between the former and the youngest and less diverse Amerindians.Assuming a dispersal of the modern humans from an African centroid, this is an indication of the relative good representation of the samples screened and the discriminative power of the marker.The multidimensional analysis performed supports this interpretation, which is in very good agreement with worldwide studies based on many more genetic markers (e.g.Cavalli-Sforza et al. 1994).However, it must be kept in mind that it is based on a single marker, and, TABLE 6 Likelihood ratio rests for step-wise mutational mechanisms related to the STR (444.41).
within intron I of The TP53 locus in ten populations
second, as noted by Harpending and Rogers (2000), measures of genetic distances of populations covering large geographic areas are often misleading.Among the fixation indexes, RsT, like genetic distances, also showed to be insensitive at major scales.A possible cause for this is the fact that three alleles (136/9, 131/8 and 126/7) are the most common in all populations and even though the size of the alleles ranged between 111/4 and 151/12, alleles other than the commonest rarely reached 10% or a higher frequency, which in turn could be the result of random fixation of alleles that can act differently on different loci.Some of these loci would have fixed different alleles in different parts of the world, as has been documented (Zerylnick et a/.1995, Deka et al. 1996Deka et al. ,1999)).It could also be the case that they preserve the same alleles in all populations as has been shown for the CSF1R locus (Deka et al. 1999).Balancing selection cannot be ruled out given the important role of TP53 in genomic stability (Janus et al. 1999), and although the marker screened is located at an intron, increased instability of mRNA from alleles with motifrepeat numbers outside the range observed could affect the normal function of the gene.In addition, some alleles might have been indirectly selected by hitchhiking if they were in cis with alleles conferring increased fitness.The latter hypothesis could be tested extending the screening to haplotypic variation.
The Filipino and the hybrid Costa Rican population deserve comments aside.The Filipino shows an intermediate diversity between the Africans and Europeans and was unexpectedly placed at the multidimensional analysis closer to the Africans rather than to the Asiatics.Cavalli-Sforza et al. (1994) found a high diversity in this group using classical markers (blood groups and blood proteins) and explained the fact as a sign of a putative multiple origin (Negrito, Malayan and Chinese).In addition, the populations of Southeast Asia stem from an early split from the group that led to the other Asiatic groups (Cavalli-Sforza et al. 1994).Both facts, fusion of different ethnic groups each originating from a time when the ancestral (African) gene diversity was not diminished by bottlenecks could explain their higher diversity and closer proximity to the Africans than to the Amerindians.Regarding the high diversity value shown by the hybrid population of Costa Rica, it also reflects its multiethnic origin.Morera et al. (2003) estimated that the Costa Rican population has 61% European -mainly Spanish-, 30% Amerindian and 9% Africans genetic ancestry.Its nearer position to the African cluster is in accordance with that estimate.In the end, admixture can be seen as a regression to the ancestral conditions.
The data obtained also permitted a statistical analysis of the main molecular mechanism producing new alleles at this marker.The test favors a model of expansion or contraction in the number of tandem repeats by single-steps, although this does not rule out multi-step mutations.This conclusion is concordant with empirical data of Brinkman et al. (1998) who analyzed 10 844 parent/child allelic transfers at 23 STRs and found that 22 out of 23 mismatches detected were single-step and only 1 a two-step.

AKNOWLEDGMENTS
We thank Omar Achi for his fine laboratory work.We also thank Carsten Wolff (Halberstadt, Germany), Ulrich Muller, Daniels Steinberger (Giessen, Germany) and Dietrich Biittner (Hamburg, Germany) for providing us the samples from Uganda, Namibia, The Philippines, Germany and Turkey.Support of Alfred Pingoud (Giessen) and Peter Lichter (Heidelberg), is also greatly acknowledged.This research was partially funded by the Universidad de Costa Rica  and by the Consejo Nacional de Investigaciones Cientificas y Tecnologicas de Costa Rica (CONICIT).
Hardy-Weinberg expectations, evaluated by a modified Fisher's exact test(Guo and Thompson 1992), and fixation indexes, R15, RsT and Rip and genetic distances were estimated according to Slatkin (1995) using the ARLEQU1N program(Schneider et al. 1997 Fig. I. Multidimensional analysis for the SIR (AAAAT)n within the intron I of the TP53 gene in ten populations.

TABLE 1
Phenotype frequencies of the STR (AAAAT).within intron 1 of the TP53 locus in Mongoloid.Numbers indicate size of the bands in base pairs (bp).The band of [41bp contains 10 (AAAAT) repeats and the 116bp 5 repeats (Hahn er al. 1995).

TABLE 3
Allele frequencies, observed and expected helennygosities, and Hardy-Weinberg equilibrium test of the SFR (AAAAT),, within intron l of the TP53 locus in Mongoloid populations: Amerindians of Cana Rica and Panama and in Filipinos