The origin of all other populations used for comparisons is detailed in Table 1.
HLA Genotyping and DNA Sequencing. Generic HLA class I (A and B) genotyping (resolution equivalent to the standard serology techniques) and high-resolution HLA class II (DRB1 and DQB1) was performed using a reverse dot-blot technique with the Automated Innolipa system (Innogenetics N.V., Zwijndrecht, Belgium). HLA-A, -B, -DRB1, and -DQB1 allele DNA sequencing was done only when indirect DNA typing yielded ambiguous results (Arnaiz-Villena et al. 1992).
Statistical Analysis. Statistical analyses were performed using Arlequin v2.000 software (Schneider et al. 2000). In summary, this program calculated HLA-A, -B, -DRB1, -DQA1, and -DQB1 allele frequencies, Hardy-Weinberg equilibrium, and the linkage disequilibrium between two alleles at two different loci. Linkage disequilibrium and the level of significance (p) for 2 x 2 comparisons were determined using the formulae of Mattiuz and coworkers (Mattiuz et al. 1970) and the 11th International Histocompatibility Workshop methodology (Imanishi et al. 1992b). In addition, the most frequent complete haplotypes were tentatively deduced using a methodology developed in the 11th International Histocompatibility Workshop. The criteria used were: (1) the two-, three-, and four-HLA-loci haplotype frequencies (Imanishi et al. 1992c; Clayton and Lonjou 1997); (2) the previously described haplotypes in other populations (Imanishi et al. 1992c; Clayton and Lonjou 1997; Arnaiz-Villena et al. 1995, 1997, 1999, 2001a, 2001b; Martinez-Laso et al. 1995, 2001; Gomez-Casado et al. 2000); and (3) haplotypes if they appeared in two or more individuals and the alternative haplotype was well defined (Imanishi et al. 1992c; Clayton and Lonjou 1997; Arnaiz-Villena et al. 1995, 1997, 1999, 2001a, 2001b; Martinez-Laso et al. 1995, 2001; Gomez-Casado et al. 2000). In order to compare allelic and haplotype HLA frequencies with other populations, the reference tables of the 11th and 12th International HLA Workshops were used (Imanishi et al. 1992c; Clayton and Lonjou 1997) (also see Table 1). The software DISPAN containing the programs GNKDST and TREEVIEW (Nei 1973, 1983) was used to construct phylogenetic trees (dendrograms) with the allelic frequencies by using the neighbor-joining (NJ) method (Saitou and Nei 1987) with genetic distances between populations (DA) (Nei 1972). Correspondence analysis in a bidimensional representation was obtained by using the VISTA v5.02 computer program (Young and Bann 1996) (http:/forrest.psych.unc.edu). Correspondence analysis consists of a geometric technique that may be used for displaying a global view of the relationships among populations according to HLA (or other) allele frequencies. This methodology is based on the allelic frequency variance among populations (similar to the classical components methodology) and on the topological display of their statistical differences.
HLA Genotyping and DNA Sequencing. Generic HLA class I (A and B) genotyping (resolution equivalent to the standard serology techniques) and high-resolution HLA class II (DRB1 and DQB1) was performed using a reverse dot-blot technique with the Automated Innolipa system (Innogenetics N.V., Zwijndrecht, Belgium). HLA-A, -B, -DRB1, and -DQB1 allele DNA sequencing was done only when indirect DNA typing yielded ambiguous results (Arnaiz-Villena et al. 1992).
Statistical Analysis. Statistical analyses were performed using Arlequin v2.000 software (Schneider et al. 2000). In summary, this program calculated HLA-A, -B, -DRB1, -DQA1, and -DQB1 allele frequencies, Hardy-Weinberg equilibrium, and the linkage disequilibrium between two alleles at two different loci. Linkage disequilibrium and the level of significance (p) for 2 x 2 comparisons were determined using the formulae of Mattiuz and coworkers (Mattiuz et al. 1970) and the 11th International Histocompatibility Workshop methodology (Imanishi et al. 1992b). In addition, the most frequent complete haplotypes were tentatively deduced using a methodology developed in the 11th International Histocompatibility Workshop. The criteria used were: (1) the two-, three-, and four-HLA-loci haplotype frequencies (Imanishi et al. 1992c; Clayton and Lonjou 1997); (2) the previously described haplotypes in other populations (Imanishi et al. 1992c; Clayton and Lonjou 1997; Arnaiz-Villena et al. 1995, 1997, 1999, 2001a, 2001b; Martinez-Laso et al. 1995, 2001; Gomez-Casado et al. 2000); and (3) haplotypes if they appeared in two or more individuals and the alternative haplotype was well defined (Imanishi et al. 1992c; Clayton and Lonjou 1997; Arnaiz-Villena et al. 1995, 1997, 1999, 2001a, 2001b; Martinez-Laso et al. 1995, 2001; Gomez-Casado et al. 2000). In order to compare allelic and haplotype HLA frequencies with other populations, the reference tables of the 11th and 12th International HLA Workshops were used (Imanishi et al. 1992c; Clayton and Lonjou 1997) (also see Table 1). The software DISPAN containing the programs GNKDST and TREEVIEW (Nei 1973, 1983) was used to construct phylogenetic trees (dendrograms) with the allelic frequencies by using the neighbor-joining (NJ) method (Saitou and Nei 1987) with genetic distances between populations (DA) (Nei 1972). Correspondence analysis in a bidimensional representation was obtained by using the VISTA v5.02 computer program (Young and Bann 1996) (http:/forrest.psych.unc.edu). Correspondence analysis consists of a geometric technique that may be used for displaying a global view of the relationships among populations according to HLA (or other) allele frequencies. This methodology is based on the allelic frequency variance among populations (similar to the classical components methodology) and on the topological display of their statistical differences.
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