ALLELE FRECUENCY DISTRIBUTIONS OF NINE LOCI STRs IN PANAMANIAN MESTIZOS M?LIDA I. N??EZ-C.*, ** TOM?S ARIAS*, ** CHYSTRIE RIGG* CARLOS RAMOS* MATTHEW J. MILLER** Resumen: La poblaci?n paname?a ha sido previamente estudiada gen?- ticamente usando marcadores tradicionales como las aloenzimas, pero hasta la fecha no se han realizado estudios que demuestren la utilidad de los short?tandem repeat markers (STR) en los protocolos para resolver casos forenses y de paternidad. La poblaci?n paname?a esta constituida por grupos poli ?tnicos y/o multirraciales, representando las tres razas principales: amerindios, negros y cauc?sicos, pero tambi?n hay un grupo representativo de personas con linaje del Este Asi?tico y Medio Oriente. Analizamos los polimorfismos gen?ticos de los siguientes loci STR: CSF1PO, TPOX, TH01, F13A01, FESFPS, VWA, D16S539, D7S820 y D13S317 en una muestra poblacional de individuos mestizos no relacionados, estos individuos resid?an en la ciu- dad de Panam?. La distribuci?n genot?pica de ocho de los nueve loci estu- diados estaba en equilibrio de Hardy?Weimberg; D16S539 mostraba un exceso de homocigotos que podr?a ser atribuido a allelic dropout. Inclusive cuando este locus es removido del an?lisis, los valores forenses (poder de discriminaci?n, matching probability y poder de exclusi?n) de estos marca- dores entre los mestizos Paname?os es extremadamente alta. En general nuestro estudio no encontramos evidencia de subestructura entre los mes- tizos Paname?os, sin embargo con las medidas para corregir el allelic dro- pout del locus D16S539, el GenePrint STR Multiplex Systems (Silver Stain CFOR, 9-10/2009-2010 107 Ciencia Forense, 9-10/2009-2010: 107-116 * Universidad de Panam?, Departamento de Gen?tica y Biolog?a Molecular, Apartado 0824, Panam?, Rep?blica de Panam?. ** Smithwonian Tropical Research Institute, Apartado 0843-03092, Balboa, Palma. Detection) es adecuado para las aplicaciones forenses y de paternidad en Panam?. Palabras claves: Panam?. Mestizo. Trihybrid modelo. Latinoam?rica. STR. Am?rica central. Abstract: The population genetics of the people of Panama have been previously explored using traditional allozyme markers, but to date, no study has examined the utility of existing short?tandem repeat marker (STR) protocols for resolving forensic and paternity cases. The Panamanian population constitutes a highly multi?racial and poly?ethnic group repre- senting three principle races: Amerindians, blacks and Caucasians, but also containing an appreciable population of people with East Asian and Middle Eastern heritage. We analyzed genetics polymorphisms for the following standard STR loci: CSF1PO, TPOX, TH01, F13A01, FESFPS, VWA, D16S539, D7S820 and D13S317 in a population sample of unrelated mixed?race (mes- tizo) individuals who resided in Panama City. Genotypic distribution was in agreement with Hardy?Weinberg expectations (HWE) for eight of nine markers; D16S539 showed an excess of homozygotes that may be attributa- ble to allelic dropout. Even when this locus is removed from analysis, the forensic value (e.g. power of discrimination, matching probability and power of exclusion) of these markers among Panamanian mestizos is extre- mely high. Overall, our study did not find evidence of substructure among Panamanian mestizos, however with measures to correct the allelic dropout of D16S539, the GenePrint STR Multiplex Systems (Silver Stain Detection) is appropriate for forensic and paternity applications in Panama. Key words: Panama. Mestizo. Trihybrid model. Latin America. STR. Cen- tral America. M?lida I. N??ez-C., Tom?s Arias, Chystrie Rigg, Carlos Ramos, Matthew J. Miller 108 CFOR, 9-10/2009-2010 INTRODUCTION The Republic of Panama is located at the southern extreme of the Cen- tral American isthmus that joins North America and South America. Pana- ma represents the narrowest point on this isthmus, and has served as a principle crossroads for interhemispheric travel and commerce for at least 500 years. The indigenous diversity of Panama is considerable; especially given the country?s size (Panama covers 75,500 km2, roughly the size of Scotland or South Carolina). Although, early archeological evidence poin- ted to repeated migration through the Panama isthmus by early Amerin- dians during the peopling of the Americas (1), genetic evidence points to two distinctive mitochondrial lineages corresponding to the two distinct indigenous language families found in Panama. European colonization of Panama began in the 16th century, when the Spanish constructed out- posts on both the Caribbean and Pacific coasts and joined them by over- land roads to facilitate the transportation of material goods, slaves and colonists between the Spanish empire in the Americas and the Old World. This event marked the first migration of African people to Panama, who came as Spanish slaves. A second migration of people of African ancestry to Panama occurred in the early 20th century when West Indians of Afri- can descent, principally men from Barbados, migrated to Panama as labo- rers on the Panama Canal. Recently, a considerable number of people of Asian and Middle Eastern origin have immigrated to Panama, as a conse- quence of the Panama Canal and Panama City?s role as a global maritime and financial center, Earlier admixture studies of Panama have concluded that the popula- tion (nearly 3 million people) best fits a trihybrid model whereby African, Amerindian, and European ancestry is shared by most of the population. Using ABO and Rh blood markers, Arias et al. (2) found in Panama that 38.7% of the genes were of African origin, while 35.9% and 25.4% of the genes were of Amerindian and European origin respectively. Other Latin American populations considered trihybrid (such as Cubans, Costa Ricans, Dominicans, and Puerto Ricans had substantially less?equal proportions of African, Amerindian, and European genes (2?3). In Panama, the term mestizo refers to a person whose with a mixture of African, Amerindian, and European ancestry; thus, most of the population is considered mestizo. Currently, the Republic of Panama applies an analysis of short?tandem repeats (STRs) for forensic investigations and paternity cases, despite any baseline information on the allelic frequency distribution of Panamanians. Furthermore, due to budgetary limitations, the current forensic protocol includes only nine of the 13 STR loci that are standard for CODIS. Given the earlier evidence of substantial admixture from three distinct genetic pools, we initiated this study to determine what those frequencies are and to evaluate the exclusionary power of the current forensic approach employed in Panama. The goal of the present study was to describe STR polymorp- Allele frecuency distributions of nine loci STRs in Panamanian mestizos CFOR, 9-10/2009-2010 109 hism in the Panamanian mestizo population, evaluate whether any apprecia- ble substructure exists within this population, and test the efficacy of the nine marker STR system currently in use in the Panamanian justice system. MATERIALS AND METHODS DNA sample were obtained from 102 unrelated mestizo Panamanians who visited the Hospital Santo Tomas, Panama City in order to donate blood. Only included in this study were native?born Panamanians with both parents having been born in Panama. We excluded from the study individuals who resided in the semi?autonomous indigenous political dis- tricts (comarcas) and/or those whose dress or phenotype suggested that they were of purely indigenous ancestry. While our sampling protocol allo- wed for any Panamanian to be include in the study, in practice, all 102 sampled individuals were born and currently live in the Panama City muni- cipality. All samples were taken between August and October, 2003. 10 ml of blood was obtained by venipuncture, collected in tubes contai- ning anticoagulant ACD, and stored in the Laboratory of Molecular Bio- logy of the University of Panama at ?20?C. DNA was extracted using stan- dard phenol?chlorophorm procedures and stored in 1X TE buffer (4). DNA was quantified and its quality and quality was evaluated by means of agarose gel electrophoresis at 0.8% in TBE 0.5X. Amplifications were performed using the GenePrint STR Multiplex Sys- tems (Silver Stain Detection) (5), which is commonly used in paternity tests and forensic work in Panama. This system consists of nine STRs loci seven of which belong of CODIS, the STRs loci that conform this system are CSF1PO, TPOX, TH01, F13A01, FESFPS, vWA, D16S539, D7S820 and D13S317. Amplifications were carried out in a Perkin?Elmer model 2400 thermalcycler. Following manufacturer?s protocols, all nine loci were amplified simultaneously in a single tube and analyzed in a single pol- yacrylamide gel lane. Before typing, we verified amplifications using agaro- se gel electrophoresis. Polyacrylamide gels were prepared and run follo- wing manufacturer?s protocols and were visualized by silver stain. Allele frequencies were calculated for each locus using the GENALEX program (6). Deviations from Hardy?Weinberg equilibrium were analyzed using three tests: chi?square (X2) tests, likelihood ratio test (G?square) (both performed in PopGene (7) and the exact test which was performed in GenePop (8). We tested for evidence of linkage disequilibrium using Arelquin (9). Empirical significance level for the exact test and the test of linkage disequilibrium were determined by 10000 permutations; results were Bonferroni corrected. The forensic analysis parameters: Power of Dis- crimination (PD), Power of Exclusion (PE), Polymorphic Information Con- tained (PIC) and Matching Probability(MP) were calculated for each locus, as well as all loci combined, were estimated using the PowerStats 1.2 (10). M?lida I. N??ez-C., Tom?s Arias, Chystrie Rigg, Carlos Ramos, Matthew J. Miller 110 CFOR, 9-10/2009-2010 We used Structure v2.2 (11) to test for evidence of sub?structure among Panamanian mestizos. Structure implements a model ?based clustering method using Bayesian Monte Carlo Markov Chain (MCMC) likelihood analysis. Priors for k, the number of sub?populations in populations, were set in the range of 1?3, since the tri?hybrid model for Panama predicts three genetic sources for mestizos. MCMC chains were run for 100,000 generations with 10,000 samples discarded as a burning. For each k value we ran the simulation 20 times to test for convergence. RESULTS AND DISCUSSION Observed allelic frequencies for the nine sampled STR loci are repor- ted in Table 1. Eight of the nine loci showed no deviations from Hardy?Weinberg equilibrium (HWE), however locus D16S539 showed a significant excess of homozygote excess, which has been reported in ear- lier studies (12), and has been attributed to allelic dropout due to a pri- mer?binding site mutation relatively common in people of African herita- ge (12). Analysis of this locus using Mircochecker (13) indicates that there is a deficiency of heterozygotes consistent with allelic dropout. Because this allele does not conform to HWE, it should not be used in forensic analysis in Panama, and thus we present cumulative forensic probabilities using the eight remaining loci (table 2) which indicate that the remaining eight loci have extremely high forensic discrimination power among Pana- manian mestizos. Levels of heterozygosity, as expected, were high among mestizos. In the Structure analysis, k=1 had the highest likelihood score. (?ln likelihood k=1: ?2689.5, ?ln likelihood k=2: ?2677.8 and ?ln likelihood k=3: ?2651.3), thus there is no evidence for substructure among mestizos in our data. Finally, we note that our sample shows uncovered rare alleles, espe- cially relative to other studies of Hispano?American populations: the rela- tively rare allele 3 for locus TH01 was observed in a single individual; this allele was previously reported in a Portuguese population (14).Furthermo- re, 1 individual in our study showed a copy of allele 22 for locus vWA, which was earlier reported from a study of Nigerians (15). CONCLUSION We conclude that the GenePrint STR Multiplex Systems (Silver Stain Detection) together provide considerable forensic discrimination poten- tial and are appropriate for use in Panama; however locus D16S539 should not be included in forensic probability calculations. Allele frecuency distributions of nine loci STRs in Panamanian mestizos CFOR, 9-10/2009-2010 111 M?lida I. N??ez-C., Tom?s Arias, Chystrie Rigg, Carlos Ramos, Matthew J. Miller 112 CFOR, 9-10/2009-2010 T ab la 1 A ll el e d is tr ib u ti o n o f 9 ST R l o ci i n a p o p u la ti o n s am p le o f P an am an ia n m es ti zo s (n =1 02 ). O b se rv ed h et er o zy go si ty ( H ), a n d s ig n if ic an ce v al u es o f te st s fo r d ev ia ti o n f ro m H ar d y? W ei n b er g eq u il ib ri u m , u si n g th re e m et h o d s: c h i? sq u ar ed t es t (X 2 ) , li ke li h o o d ?r at io t es t (G 2 ) , an d F is h er ?s e xa ct t es t, a ft er B o n fe rr o n i co rr ec ti o n s. A ll el e C SF 1P O T P O X T H O 1 F 13 A 01 F E SF P S V W A D 16 S5 39 D 7S 82 0 D 13 S3 17 3 ? ? 0. 00 5 ? ? ? ? ? ? 3. 2 ? ? ? 0. 09 8 ? ? ? ? ? 4 ? ? ? 0. 23 5 ? ? ? ? ? 5 ? ? 0. 01 0 0. 23 0 ? ? 0. 01 0 ? ? 6 ? 0. 01 0 0. 42 2 0. 17 6 ? ? 0. 01 5 ? ? 7 0. 02 5 0. 01 5 0. 23 5 0. 17 6 ? ? 0. 05 4 0. 00 5 0. 00 5 8 0. 00 5 0. 44 1 0. 12 7 0. 03 4 0. 05 4 ? 0. 08 3 0. 11 8 0. 04 9 9 0. 03 4 0. 06 4 0. 11 8 ? 0. 02 0 ? 0. 19 1 0. 10 3 0. 12 7 9. 3 ? ? 0. 04 4 ? ? ? ? ? ? 10 0. 19 6 0. 07 8 0. 03 9 0. 00 5 0. 15 7 ? 0. 19 1 0. 25 5 0. 06 9 11 0. 13 4 0. 26 0 ? 0. 00 5 0. 42 2 ? 0. 24 5 0. 31 4 0. 20 1 12 0. 33 8 0. 12 7 ? 0. 01 0 0. 26 5 ? 0. 14 7 0. 18 6 0. 26 5 13 0. 07 8 0. 00 5 ? 0. 01 5 0. 08 3 0. 01 0 0. 06 4 0. 02 0 0. 21 1 14 0. 01 0 ? ? 0. 00 5 ? 0. 04 9 ? ? 0. 06 9 15 ? ? ? 0. 01 0 ? 0. 12 3 ? ? 0. 00 5 16 ? ? ? ? ? 0. 32 4 ? ? ? 17 ? ? ? ? ? 0. 25 0 ? ? ? 18 ? ? ? ? ? 0. 15 7 ? ? ? 19 ? ? ? ? ? 0. 07 4 ? ? ? 20 ? ? ? ? ? 0. 01 0 ? ? ? 21 ? ? ? ? ? ? ? ? ? 22 ? ? ? ? ? 0. 00 5 ? ? ? H 71 .6 % 70 .6 % 67 .6 % 79 .4 % 69 .6 % 74 .4 % 69 .6 % 81 .4 % 83 .3 % X 2 0. 25 5 0. 98 0 0. 00 6 0. 74 1 0. 02 7 0. 51 2 0. 00 0* 0. 27 3 0. 00 6 G 2 0. 79 5 0. 97 5 0. 02 7 0. 99 7 0. 08 4 0. 64 7 0. 00 0* 0. 44 2 0. 11 9 E xa ct ?t es t 0. 03 8 0. 22 9 0. 33 9 0. 33 6 0. 05 1 0. 05 5 0. 00 0* 0. 53 3 0. 29 1 * si gn if ic an t P v al u e af te r B o n fe rr o n i co rr ec ti o n s (a = 0. 00 5) . Allele frecuency distributions of nine loci STRs in Panamanian mestizos CFOR, 9-10/2009-2010 113 Tabla 2 Forensic statistics parameters for eight of nine loci included in the GenePrint system. Matching Polymorphism Locus Matching Probability Power of Information Power of Probability Expressed Discrimination Content Exclusion as 1 in? CSF1PO 0.108 9.2 0.892 0.70 0.453 TPOX 0.126 8.0 0.874 0.67 0.437 TH01 0.111 9.0 0.889 0.70 0.393 F13A01 0.132 16.1 0.938 0.79 0.588 FESFPS 0.132 7.6 0.868 0.67 0.422 vWA 0.084 11.9 0.916 0.75 0.485 D7S820 0.099 10.1 0.901 0.74 0.625 D13S317 0.072 14.0 0.928 0.79 0.662 8 loci 1.9X10?7 1.36X108 0.999 0.999 0.997 (cumulative) Figure 1. Results of Structure MCMC simulations assuming k number of subpopulations among Panamanian mestizos. Each horizontal bar repre- sents a single individual in the study, and the colors represent posterior probability values of assigning that individual to a given subpopulation. Note that when k > 1, no individual has high posterior value of being assig- ned to a single subpopulation, furthermore the model with k=1 had the highest ?ln likelihood, indicating no substructure among our sample of Panamanian mestizos. 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