¹Department of Physiology, Instituto Nacional de Cardiologia "Ignacio Chávez", México City, México

²Department of Hematology, Instituto Nacional de Cardiologia "Ignacio Chávez", México City, México

³Molecular Biology and Immunogenetics Laboratory, Infectious Diseases Unit, Instituto Nacional de Enfermedades Respiratorias, México City, México

⁴Department of Immunology and Rheumatology, Instituto Nacional de Ciencias Medicas y Nutrición Salvador Zubirán, México City, México

Correspondence to: Dr G Vargas-Alarcón, Department of Physiology, Instituto Nacional de Cardiología Ignacio Chávez. Juan Badiano No. 1, Tlalpan 14080, México D.F, México. E-mail: gvargas63@yahoo.com

This work was supported in part by grants from the Consejo Nacional de Ciencia y Technología. México D.F., México.

Low molecular weight polypeptide (LMP) genes are located within the major histocompatibility complex and have been associated with autoimmune diseases such as ankylosing spondylitis. In order to define the distribution of LMP genes in Mexican populations, the LMP2 and LMP7 polymorphism was analyzed in 312 Mexican individuals (95 Mexican Mestizos, 48 Nahuas, 56 Mazatecans, 50 Teenek, and 63 Mayos) belonging to different ethnic groups. In Mexican populations both Mestizos and Amerindians presented similar distribution of LMP2 and LMP7 polymorphisms, except Nahuas and Mayos who presented the higher frequencies of LMP2-H/H and the lowest frequencies of LMP2-H/R genotypes (P < 0.05 when compared with Mexican Mestizos). The LMP7-K/K genotype was absent in Nahuas, Teenek and Mayos and only one Mazatecan individual presented this genotype. Differences with other populations were found in Mexicans. An increased frequency of LMP2-H and a decreased frequency of LMP2-R alleles were observed in Mexican Amerindians (Nahuas and Mayos) when compared with Brazilian Amerindians (Kaingang and Guarani) and Caucasians (Spaniards) (P < 0.05). All Mexican populations (Mestizos and Amerindians) presented an increased frequency of LMP7-Q allele and a decreased frequency of LMP7-K allele when compared to Brazilian Amerindians (Kaingang), Caucasians (United States) and Asian (Japan) populations (P < 0.05). Genetic distances showed that Mexican Mestizos have an important relation with Spaniards and with all Mexican Amerindians. The present data corroborate the influence of Spaniard and Amerindian genes in the Mexican Mestizo population and could help to define the true significance of LMP polymorphism as genetic and evolutive marker in the Amerindian populations.

Genes and Immunity (2002) 3, 373-377. doi:10.1038/sj.gene.6363855

low molecular weight polypeptides; major histocompatibility complex; polymorphism; Mexican ethnic groups

Introduction

Low molecular weight polypeptides (LMP) genes are located within the major histocompatibility complex (MHC) class II region.^1,2,3,4 LMP genes are polymorphic and its products constitute two subunits of the proteosome complex involved in the degradation of cytosolic proteins and generation of antigenic peptides.^4,5

Due to the participation of the LMP products in the antigen presentation, where they may play an important restrictive role, they are attractive candidates as additional autoimmune disease susceptibility factors. Diseases studied in relation to the possible participation of these genes as genetic markers include rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (IDDM) and ankylosing spondylitis (AS).^6,7,8 However, several reports show lack of association or conclude that this association might be secondary to linkage disequilibrium with HLA genes primary associated with the disease.^9,10,11 Studies of associations with diseases across ethnic groups are important in removing the confounding effects of linkage disequilibrium and are helpful to localize the actual disease-conferring alleles. For this reason population genetics studies can contribute to understand the possible role of the LMP genes as genetic and evolutive markers. Population studies of LPM polymorphism are rare and the majority refers mostly to Caucasian and Oriental populations.^6,8,9,12 In an effort to define genetic admixture in Mexican Mestizos, this ethnic group has been characterized by using genetic markers from several chromosomes. Results from these studies have shown a proportion of 56% Amerindian genes, 40% Caucasian genes, and 4% black genes.^13,14,15 The aim of the present work was to study the LMP allele and genotype distribution in Mexican populations and establish the admixture between them and other populations using this polymorphism.

Results

Genotype and allele frequencies of LMP2 and LMP7 in different Mexican populations (Mestizos and Amerindians) are shown in Table 1. In all samples the observed and expected frequencies are in Hardy-Weinberg equilibrium. The most common LMP2 genotype was homozygous R/R (51.5% in Mexican Mestizos, 58.3% in Nahuas, 62.5% in Mazatecans, 58.0% in Teenek, and 58.7% in Mayos) and the most frequent allele was R (73.1% in Mexican Mestizos, 65.6% in Nahuas, 76.7% in Mazatecans, 72.0% in Teenek, and 65.1% in Mayos). On the other hand, the LMP7-Q/Q genotype (85.2% in Mexican Mestizos, 83.3% in Nahuas, 92.3% in Mazatecans, 82.0% in Teenek, and 74.6% in Mayos) and LMP7-Q allele (89.4% in Mexican Mestizos, 91.6% in Nahuas, 94.2% in Mazatecans, 91.0% in Teenek, and 87.3% in Mayos) were the most frequent LMP7 genotype and allele detected in all populations. The K/K genotype of LMP7 was absent in Nahuas, Teenek and Mayos, and only one Mazatecan individual presented this genotype. Mexican populations both Mestizo and Amerindians presented similar distribution of LMP2 and LMP7 polymorphisms, except Nahuas and Mayos who presented the higher frequencies of LMP2-H/H and the lowest frequencies of LMP2-H/R genotypes (P < 0.05 when compared with Mexican Mestizos). Also, Mayos presented the highest frequency of LMP7 Q/K genotype (P < 0.05 when compared with Mexican Mestizos and Mazatecans). LMP2 and LMP7 allele frequencies in Mexican populations were compared with those previously described in other ethnic groups (Table 2). An increased frequency of LMP2-H and a decreased frequency of LMP2-R alleles were observed in Mexican Amerindians (Nahuas and Mayos) when compared with Brazilian Amerindians (Kaingang and Guarani) and Caucasians (Spaniards) (P < 0.05). All Mexican populations (Mestizos and Amerindians) presented an increased frequency of LMP7-Q allele and a decreased frequency of LMP7-K allele when compared with Brazilian Amerindian (Kaingang), Caucasian (United States) and Asian (Japan) populations (P < 0.05).

Genetic distances calculated from LMP allele frequencies obtained in Mexicans (Mestizos, Nahuas, Mazatecan, Teenek and Mayos) and other previously described in several Amerindian (Kaingang and Guarani), Caucasian (United States and Spain) and Asian (Japan) populations are shown in Table 3. As it is shown, Mexican Mestizos show an important relationship with Spaniards and with all Mexican Amerindian groups. Mazatecans are related with Mayos (DA = 0.19), Mestizos (DA = 0.27) and Spaniards (DA = 0.31). Nahuas are related with Mayos (DA = 0.12), Teenek (DA = 0.12) and Mestizos (DA = 0.20). On the other hand, Mayos show relation with Mestizos (DA = 0.02), Nahuas (DA = 0.12) and Mazatecans (DA = 0.19). Finally, Teenek are related with Nahuas (DA = 0.12), Mestizos (DA = 0.22) and Mayos (DA = 0.23).

Discussion

LMP genes are located within the MHC class II region and its products have an important role in antigen presentation by class I MHC molecules.^16,17,18,19 They are polymorphic and the possible functional implications of this genetic variation are at the moment unclear. In addition, polymorphisms in LMP2 and LMP7 have not been extensively studied in human populations. Thus, the present study describe the allele and genotype LMP frequencies in Mexican Mestizos and in four Mexican Amerindian populations (Nahuas, Mazatecans, Teenek and Mayos) belonging to different linguistic trunks (data obtained from the Instituto Nacional Indigenista, México). The frequencies were compared among them and with other previously described frequencies in other populations.^6,12,20 LMP2 and LMP7 allele and genotype frequencies distribution were similar among Mexicans, except on Nahuas and Mayos that bear the highest frequencies of LMP2-H/H genotype when compared with Mexican Mestizos, Mazatecans and Teenek populations. Despite the similarities among the groups, these data suggest that the differences in frequencies of LMP2 genotypes could be helpful in distinguishing each of these populations particularly when they belong to different linguistic trunks. Mexican populations present the lowest frequencies worldwide of LMP7-K allele and in consequence the LMP7-K/K genotype is absent in these Amerindian groups (only one Mazatecan individual presents this genotype). Recently, a previous study reported this low LMP7-K allele frequency in an Amerindian tribe of Brazil (Guarani); however, other tribes analyzed in this study showed a high frequency of this allele (Kaingang).¹² The low frequency of LMP7-K allele in Native American groups suggests that this allele probably was absent in humans from northern Asia who settled in the Arctic and populated the Americas more than 25000 years before the present and that it has been acquired more recently by genetic admixture.²¹

In order to define genetic admixture in Mexican Mestizos, this ethnic group has been characterized by using genetic markers from several chromosomes. Results from these studies have shown a proportion of 56% of Amerindian genes, 40% of Caucasian genes and 4% of black genes in this population.^13,14,15 Genetic distances calculated from LMP allele frequencies show that Mexican Mestizos are related to Spaniards and Mexican Amerindians. These data corroborate the previously admixture results reported using other genetic markers and confirm the presence of Spaniard genes (who came to America during the 16th century) in the Mexican Mestizo population.

Genetic distances showed important differences between Amerindians (Mexican and Brazilian). Brazilian Amerindians (Kaingang and Guarani) show the lowest LMP2-H allele frequency whereas Kaingang present the lowest LMP7-Q allele frequencies when compared with Mexican Amerindians. Genetic distance estimation, based on HLA and other polymorphisms, revealed that Kaingang and Guarani are the most divergent among South American groups.²² A previous study where HLA polymorphism in Mexican Mazatecans were compared with 59 different populations (15 Amerindian groups) showed an important relationship between this Mexican Amerindian group and the rest of Amerindians including those of South America.²³ This suggests that Kaingang and Guarani distinguish themselves from the rest of Amerindians (North and South Amerindians) and this difference probable derives from subsequent founder effects and other forms of random genetic drift that could have been acted over these populations.

Due to the important participation of LMP gene products in the antigen presentation by class I MHC molecules, their possible role as genetic markers for autoimmune diseases has been proposed.^{6,7,8,9,10,11} Thus, the detailed distribution of LMP alleles in Mexican populations could help define the participation of this polymorphism in the genetic susceptibility for certain diseases. Few data related to the participation of LMP polymorphism in the genetic susceptibility to develop autoimmune disease have been reported. Some of them include diseases such as ankylosing spondylitis (AS), rheumatoid arthritis (RA) and insulin-dependent diabetes mellitus (IDDM).^6,7,8 However, positive association has only been detected in patients with AS.⁸ In the Mexican population the LMP2-H allele has been associated with acute anterior uveitis (AAU) in AS patients.²⁴ If this association is relevant, then the low frequency of this allele in Mexican populations could explain the low prevalence of AAU in Mexican patients with AS.

In summary, the present study shows the relationship between Mexican populations using a genetic marker that has not been analyzed previously. These data corroborate the influence of Spaniard and Amerindian genes in the Mexican Mestizo population. Since LMP alleles are known to be associated with the development of the disease as a positive risk factor, knowledge of the distribution of these alleles in Mexican populations may also be helpful in the detection of possible susceptibility genes involved in LMP-associated diseases.

Methods

The study included 312 healthy unrelated individuals obtained from five Mexican populations. A panel of 95 healthy unrelated Mexican Mestizo individuals was included. All individuals from this group were living in Mexico City, so they could be considered representative for most of Mexico inhabitants, since Mexico City has been an important immigration geographical site. Each individual was asked about his birthplace as well as that of his parents and maternal and paternal grandparents. We considered as Mexican Mestizos only those individuals who for two generations, including their own, had been born in Mexico. A Mexican Mestizo is defined as someone born in Mexico who is a descendant of the original autochthonous inhabitants of the region and of individuals, mainly Spaniards, of Caucasian and/or black origin, who came to America during the 16th century. A sample of 56 healthy, unrelated individuals from Mazatecan ethnic group, living in 'Huautla de Jimenez' and 'San Mateo Yoloxochitl' villages in the Mexican State of Oaxaca was included in the study. Mazatecans who inhabit northern Oaxaca and some areas of the Veracruz State are linguistically classified within Olmeca-Otomangue group, Otomiano-Mixteco subgroup and Popoloca family.²⁵ Forty-eight individuals belonging to the ethnic group included in the Macro-Nahua linguistic family (Nahuas located in the Mexico State), 63 belonging to the Macro-Yuma linguistic family (Mayos located in the Northeast of Mexico) and 50 individuals belonging to a group linguistically unclassified (Teenek) located in the Huasteca region of the San Luis Potosi State, were studied as well.

PCR amplification

Genomic DNA from whole blood containing EDTA was extracted by standard techniques. In order to obtain the sequence that includes the polymorphic sites, the previously described primers, LMP2X: 5' CTT GAA CCA GGG AGG CGA AGT TTG 3'; and LMP2Y: 5' CAG CGT AAC CGA AGA GTG CAT AGT 3' for LMP-2 and LMP7X: 5' CGG ACA GAT CTC TGG GTG CT 3'; and LMP7Y 5' CTT CCC TAC TGC CCC AAG CT 3' for LMP7 were used to amplify genomic DNA by using the polymerase chain reaction (PCR).⁶ PCR amplification was performed on 0.25 g of genomic DNA using 1 unit of Taq DNA polymerase (Promega, Madison, WI, USA), 200 M each dNTP, 2 mM MgCl₂, 1´ PCR buffer and 25 pmoles of each primer in a 25 l volume. Amplifications were carried out in a Perkin Elmer model 9700 (Foster City, CA, USA) thermocycler and the cycling conditions were as follows: 30 cycles of 94°C 60 s, 62°C 45 s, and 72°C 60 s for LMP7 and 30 cycles of 94°C 60 s, 57°C 50 s, and 72°C 60 s for LMP2.

Restriction of amplified products

The LMP2 polymorphism was detected by RFLP using the CfoI digestion for LMP2*R and Alw21I for LMP2*H, whereas LMP7 polymorphism was defined by using the BsmI to identified LMP7*Q allele and HindIII for LMP7*K as previously described.⁶ The fragments obtained were electrophoresed in a 10% polyacrylamide no denaturing gel and visualized by silver staining. The sizes of fragments were estimated by comparison with previously known size markers.

Statistical analysis

Allele and genotype frequencies of LMP2 and LMP7 were obtained by direct counting. Also, Hardy-Weinberg equilibrium was tested using the ARLEQUIN program. Allele frequencies obtained in Mexican populations were compared with those reported in other populations using Mantel-Haenszel chi-square. Fisher's exact test was used if the number in any cell of the 2 ´ 2 contingency table was less than five. Genetic distances (DA)²⁶ were calculated from LMP allele frequencies using the DISPAN software.

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Table 1 Genotype and allele frequencies of LMP2 and LMP7 in Mexican populations

Table 2 LMP allele frequencies in various populations including the Mexicans

Table 3 Genetic distances (DA) between Mexicans and other populations (´10²) obtained by using LMP2 and LMP7 allele frequencies