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Absence of the HLA-G*0113N allele in Amerindian populations from the Brazilian Amazon region
Human Immunology 71 (2010) 428 – 431
Contents lists available at ScienceDirect
Absence of the HLA-G*0113N allele in Amerindian populations from the Brazilian Amazon region Celso T. Mendes-Junior a,*, Erick C. Castelli b, Philippe Moreau c, Aguinaldo L. SimÖes d, Eduardo A. Donadi b a
Departamento de Quìmica, Faculdade de Filosofia, Ciéncias e Letras de Ribeirâo Preto, Universidade de Sâo Paulo, 14040-901, Ribeirâo Preto-SP, Brazil Divisâo de Imunologia Clìnica, Departamento de Clìnica Mèdica, Universidade de Sâo Paulo, 14048-900, Ribeirâo Preto-SP, Brazil c Commissariat Þ l’Energie Atomique/DSV/I2BM/Service de Recherches en Hèmato-Immunologie, IUH, Hópital Saint-Louis, 75010 Paris, France d Departamento de Genètica, Faculdade de Medicina de Ribeirâo Preto, Universidade de Sâo Paulo, 14049-900, Ribeirâo Preto-SP, Brazil b
A R T I C L E
I N F O
Article history: Received 13 August 2009 Accepted 13 January 2010 Available online 4 February 2010
Keywords: Amerindians HLA-G Natural selection Null allele South America
A B S T R A C T
The HLA-G gene is predominantly expressed at the maternal-fetal interface and has been associated with maternal-fetal tolerance. The HLA-G*0113N is a null allele defined by the insertion of a premature stop codon at exon 2, observed in a single Ghanaian individual. Likewise the G*0105N allele, the occurrence of the HLA-G*0113N in a population from an area with high pathogen load suggests that the reduced HLA-G expression in G*0113N heterozygous placentas could improve the intrauterine defense against infections. The presence of the G*0113N allele here was investigated in 150 Amerindians from five isolated tribes that inhabit the Central Amazon and in 295 admixed individuals from the State of SÄo Paulo, Southeastern Brazil, previously genotyped for HLA-G. No copy of the G*0113N null allele was found in both population samples by exon 2 sequence-based analysis, reinforcing its restricted occurrence in Africa. 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The human leukocyte antigen G (HLA-G) gene is predominantly expressed at the maternal-fetal interface and has primarily been associated with maternal-fetal tolerance, enabling fetuses to survive unharmed in a genetically foreign host during successful pregnancies [1–3]. HLA-G is involved on the inhibition of cytotoxic T lymphocyte and natural killer cytolytic functions, preventing the proliferation of CD4⫹ T cells, and tolerizing dendritic cells through interactions with specific killer-cell immunoglobulin-like receptors and immunoglobulin-like transcript receptors expressed on these cells. Because of these interactions, HLA-G has been well recognized as a tolerogenic molecule [1]. The expression of HLA-G is not usually detected in normal tissues. In nonpathological conditions, HLA-G has been detected in the placenta, thymus, cornea, hematopoietic cells, proximal nail matrix, and pancreas. In pathologic situations, HLA-G expression has been observed in several tumors, viral infections, autoimmune diseases, and grafted tissues [4]. The HLA-G locus may generate multiple protein isoforms by alternative splicing of a single mRNA, giving rise to four membranebound (HLA-G1 to ⫺G4) and three soluble isoforms (HLA-G5 to ⫺G7) [1]. Compared with classical class I HLA loci, HLA-G exhibits a reduced coding-region genetic diversity, as has been observed in * Corresponding author. E-mail address: [email protected] (C.T. Mendes-Junior).
different populations [5–9]. Such lower degree of polymorphism of HLA-G may ensure the survival of the allogeneic fetus [2]. In fact, signatures of natural selection on the promoter and coding regions of the HLA-G gene, as well as in its vicinities, have been reported in worldwide populations [10 –13]. To date, 45 HLA-G alleles have been officially recognized, presenting sequence variations in coding and noncoding regions (IMGT ⫺ International Immunogenetics Information System, November 2009, http://www.ebi.ac.uk/imgt/ hla/stats.html). HLA-G alleles have been assigned to 17 distinct allele groups, generating 15 different membrane-bound proteins and two truncated nonfunctional HLA-G1 protein encoded by two null alleles. The first one, G*0105N, is defined by a ⌬C deletion at the last base of codon 129 or the first base of codon 130 in exon 3 that causes a shift in the reading frame [8,9]. The G*0105N allele is associated with an incomplete formation of the HLA-G1, ⫺G4, and ⫺G5 isoforms, and the expression of normal ⫺G2, ⫺G3, and ⫺G7 isoforms [1,9]. The second one, G*0113N [14], is defined by a C ¡ T transition at the first base of codon 54 (Fig. 1) in exon 2 (alpha-1 domain) that determines the presence of a premature TAG stop codon; such allele determines an incomplete formation of all HLA-G membrane-bound and soluble isoforms, resulting in presumably nonfunctional proteins [14]. Although the G*0105N allele frequencies vary across different geographic regions, being absent in Amerindians from the Brazilian Amazon, and ranging from 0% to 6.1% (Spain) in Europe, from 0% to 20.0% (Iran) in Asia, and from 4.8% (Ghana) to 11.1% (Zimbabwe) in
0198-8859/10/$32.00 - see front matter 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2010.01.014
C.T. Mendes-Junior et al. / Human Immunology 71 (2010) 428 – 431
429
ity decline, which is happening even in the least developed countries, in which the average total fertility rate is now 5.2 children per woman. However, demographic data from six of the villages (from Katukina, Kaxinàwa, and Tikùna tribes) considered in the present study present a surprising average of 8.0 children per woman that reached the end of the reproductive period (ranging from 6.7 ⫾ 0.6 to 10.0 ⫾ 1.5) [24,25]. This suggests that the South-American Indians present unique cultural, environmental, demographic, and genetic features that render them special interest regarding the implications of HLA-G polymorphisms on fertility. 2. Subjects and methods
Fig. 1. Alignment of HLA-G alleles determined by nucleotide substitutions at exon 2 of such gene (complete sequence alignment of 44 HLA-G alleles identified by November 2009 – database release 2.27.1–is available at the Anthony Nolan Research Institute homepage, http://hla.alleles.org/data/hla-g.html).
sub-Saharan African populations (reviewed by [15–17]), the recently identified G*0113N was found in a single individual in a Ghanaian cohort of 100 samples [14]. Given the putative role of HLA-G in placental development, it has been proposed that the G*0105N allele is maintained in high frequency in some populations by non-neutral evolutionary forces [10]. The highest frequencies of G*0105N in geographic areas with a historically high pathogen load suggest that intrauterine pathogens may act as selective agents, increasing survival of heterozygous fetuses carrying the G*0105N allele in infected pregnancies (balancing selection). In this case, the reduced HLA-G1 expression in G*0105N heterozygous placentas may result in an overall increase in the number of T cells available in the uterus and reverse the inhibition of NK cells, improving the intrauterine defense against infections [10,18]. The survival of healthy individuals homozygous for the G*0105N allele [7,18,19] that do not express the G1, G4, and G5 soluble isoforms suggest that such isoforms are not essential for fetal survival in uncomplicated pregnancies [10,20]. Given that G*0113N transcripts do not result in any functional protein isoforms, the observation of a healthy homozygous individual for the G*0113N allele would be unexpected. Despite the different patterns of expression and functional consequences of G*0113N and G*0105N alleles, the evolutionary hypothesis proposed [10] for G*0105N might be suitable for G*0113N. However, the fact that the G*0113N allele was so far observed in a single Ghanaian individual [14] suggests a new mutational event that occurred in that individual or his/her family (private allele), keeping itself apart from the action of natural selection. By contrast, it is also possible that previous researches failed to identify such allele in worldwide populations since it was unknown. To further explore the population dynamics of G*0113N null allele, we investigated its occurrence among South American indigenous populations, in whom pregnancies have been rarely monitorized by specialized medical staff. In South America, particularly in the Amazon Rainforest, there are many pathogens [21] capable of triggering infections that may cause abortions and stillbirths [22,23]. Such intrauterine infections could have stronger effects on fetuses homozygous for functionally normal alleles than on heterozygous individuals carrying null alleles. Amerindian isolated populations exhibit high birth rates. According to a report prepared by the Department of Economic and Social Affairs of the United Nations (World Population Aging: 1950 – 2050, available at http://www.un.org/esa/population/publications/ worldageing19502050/), there is a worldwide trend toward fertil-
The population samples analyzed here were obtained from indigenous Brazilian populations visited in 1976 during the Alpha Helix expedition [26,27]. The 150 chosen Amerindians, without direct kinship, belonged to ten villages pertaining to five tribes (Katukina, Kaxinàwa, Marùbo, Tikùna, and Yaminawa) that inhabit the Central Amazon. Admixture levels with non-Amerindians were estimated to be definitely lower than 5% [26 –28]. Detailed descriptions of these populations, such as geographic distribution, linguistics, and demographic information, can be found elsewhere [26 –28]. Blood samples were processed and DNA was extracted as described elsewhere [12,15,29]. The G*0113N allele is defined by a C ¡ T transition at the first base of codon 54 in exon 2 that determines the presence of a premature TAG stop codon. The presence of such deletion was investigated according to a previously described method [6,30]: exon 2 was individually amplified yielding polymerase chain reaction (PCR) products of 361 bp, which were directly sequenced. The obtained sequences were aligned with the genomic sequences of the official alleles (recognized by the IMGT), and the existence of the G*0113N allele was carefully verified. Additionally, sequence data from 295 admixed individuals from the State of SÄo Paulo, Southeastern Brazil, previously genotyped for HLA-G exon two [6,30], were reexamined for the G*0113N mutation. 3. Results and discussion Since the first observations that HLA-G variability may have been shaped by non-neutral evolutionary forces, particularly balancing selection [10 –13], we have been studying HLA-G polymorphisms in Amerindian populations from the Brazilian Amazon [12,15] to evaluate and obtain some knowledge regarding such process in isolated South American populations. In a first attempt, the analysis of the 14-bp insertion/deletion polymorphism at the HLA-G 3= untranslated region (3= UTR) revealed a relatively low insertion frequency (38.54%) in 384 Amerindians and a trend toward balancing selection at this locus [12]. In a second effort, no copy of the G*0105N null allele was found in 143 Amerindians; such absence may be due to demographic or selective factors apart from balancing selection [15]. In the present study, the G*0113N null allele was not found in the Amerindian sample of 150 individuals (300 alleles). One may infer that the frequency of this allele is not higher than 1% in the Amerindian population, since otherwise, according to the binomial distribution, the probability of such finding (i.e., not observing the given allele in a sample of 300 alleles given that it exists in the population that originated the sampled individuals) would be lower than 5%. The lack of the null allele in the Amazonian Indians may be due to its probable absence in the migratory waves that originated from the gene pool of South American Indians. The support for this conclusion was derived from the fact that the G*0113N allele was observed in a single African individual so far [14]. Another possibility is that natural selection could have acted against HLA-G null alleles (G*0105N and G*0113N) in South American
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C.T. Mendes-Junior et al. / Human Immunology 71 (2010) 428 – 431
Indians, suggesting that membrane-bound isoforms are necessary for fetal defense against pathogens in the Amazonian environment. To further explore the existence of the G*0113N allele in South America, the reexamination of HLA-G exon 2 sequence data [6,30] from 295 admixed individuals (590 alleles) sampled from different places in the State of SÄo Paulo, Southeastern Brazil, confirmed the absence of such null allele in a highly diverse population. Following the same rationale described earlier in the text, it may be inferred that the frequency of the G*0113N allele is not higher than 0.5% in the Brazilian urban population. It should be emphasized that Brazilians represent one of the most heterogeneous populations in the world, which is the result of five centuries of genetic admixture between people from three continents: the European colonizers mainly represented by the Portuguese, the African slaves, and the autochthonous Amerindians [31]. Therefore, the Brazilian urban population has presented the largest HLA-G variability already detected [6,30,32]. Therefore, the present observation suggests that G*0113N is a Ghanaian private mutation rather than a worldwide polymorphic allele. Population genetics theory states that evolution is a dynamic process. Sometimes selection can vary over time, with different alleles being favored at different time intervals, as selective regimes change [33]. The great majority of random mutations that arise are neutral or deleterious. However, advantageous mutations can also arise by chance, allowing a higher fitness for a heterozygous individual compared with a homozygote. Much effort has been devoted to evaluate the action of balancing selection in the human genome [34]. Throughout evolutionary history, HLA-G expression levels may have evolved resulting in a fine balance between optimal levels of expression from fetal to adult life [13], allowing both fetal tolerance and appropriate intrauterine defense against pathogens: low HLA-G expression could stimulate recurrent abortions, whereas high HLA-G expression could provide susceptibility to intrauterine infection by pathogens. A high HLA-G expression would be acceptable in many geographic regions, but in areas where many intrauterine pathogens exist, both tolerance and defense would be desired. According to this hypothesis [10,13], in such specific environments a new mutation that would disrupt HLA-G expression could be advantageous in heterozygosis, providing intermediate HLA-G levels. Signatures of balancing selection on the promoter [13], coding [10], and 3= UTR [12] regions of the HLA-G gene, as well as the observation that variation in the HLA-G promoter region influences transcription rates and that increased expression of HLA-G may be deleterious in some pregnancies [35], support the heterozygous advantage hypothesis. It is possible that in pregnancies with subclinical infection, increased levels of HLA-G may compromise maternal cellular immunity to invading pathogens [35]. In conclusion, the G*0113N allele has not been observed in the autochtonous or admixed South-American populations considered herein. However, its absence in Asia and Europe, as well as its limited occurrence in Africa, should be confirmed by the reexamination of previously generated HLA-G exon 2 sequence data or by the evaluation of individuals from other population samples from such geographic areas. This would contribute to elucidate the geographic distribution and population dynamics of the G*0113N allele and to further explore the role of natural selection over the HLA-G gene. Acknowledgments The authors thank Dr. Simone Kashima, Sandra Rodrigues, Rochele Azevedo, Evandra Strazza Rodrigues and Adriana Marques for invaluable technical help. This study was supported by CNPq (Conselho Nacional de Desenvolvimento CientÎfico e TecnolÔgico - Brazil) Grant 475670/2007-08, CAPES (CoordenaÈÄo de AperfeiÈoamento de Pessoal de NÎvel Superior), FAPESP (FundaÈÄo de Amparo Á Pesquisa do Estado de SÄo Paulo) and FAEPA (FundaÈÄo de Apoio ao Ensino, Pesquisa e AssistËncia do HCFMRP-USP). C.T.M.J. was sup-
ported by a post-doctoral fellowship from CNPq/Brazil (150996/ 2005-5) and latter from FAPESP/Brazil (07/58391-4), and by a travel grant to Paris, France, from FAPESP/Brazil (2009/06191-7). E.C.C. is supported by a post-doctoral fellowship (07/58420-4) from FAPESP/Brazil. References [1] Carosella ED, Favier B, Rouas-Freiss N, Moreau P, Lemaoult J. Beyond the increasing complexity of the immunomodulatory HLA-G molecule. Blood 2008;111:4862–70. [2] Ober C, Aldrich CL, Chervoneva I, Billstrand C, Rahimov F, Gray HL, et al. Variation in the HLA-G promoter region influences miscarriage rates. Am J Hum Genet 2003;72:1425–35. [3] Hviid TV. HLA-G in human reproduction: Aspects of genetics, function and pregnancy complications. Hum Reprod Update 2006;12:209 –32. [4] Carosella ED, Moreau P, Lemaoult J, Rouas-Freiss N. HLA-G: From biology to clinical benefits. Trends Immunol 2008;29:125–32. [5] Abbas A, Tripathi P, Naik S, Agrawal S. Analysis of human leukocyte antigen (HLA)-G polymorphism in normal women and in women with recurrent spontaneous abortions. Eur J Immunogenet 2004;31:275– 8. [6] Castelli EC, Mendes-Junior CT, Donadi EA. HLA-G alleles and HLA-G 14 bp polymorphisms in a Brazilian population. Tissue Antigens 2007;70:62– 8. [7] Matte C, Lacaille J, Zijenah L, Ward B, Roger M. HLA-G and HLA-E polymorphisms in an indigenous African population. The ZVITAMBO Study Group. Hum Immunol 2000;61:1150 – 6. [8] Ober C, Aldrich CL. HLA-G polymorphisms: Neutral evolution or novel function? J Reprod Immunol 1997;36:1–21. [9] Suarez MB, Morales P, Castro MJ, Fernandez V, Varela P, Alvarez M, et al. A new HLA-G allele (HLA-G*0105N) and its distribution in the Spanish population. Immunogenetics 1997;45:464 –5. [10] Aldrich C, Wambebe C, Odama L, Di Rienzo A, Ober C. Linkage disequilibrium and age estimates of a deletion polymorphism (1597DeltaC) in HLA-G suggest non-neutral evolution. Hum Immunol 2002;63:405–12. [11] McEvoy BP, Montgomery GW, McRae AF, Ripatti S, Perola M, Spector TD, et al. Geographical structure and differential natural selection among North European populations. Genome Res 2009;19:804 –14. [12] Mendes-Junior CT, Castelli EC, SimÖes RT, SimÖes AL, Donadi EA. HLA-G 14-bp polymorphism at exon 8 in Amerindian populations from the Brazilian Amazon. Tissue Antigens 2007;69:255– 60. [13] Tan Z, Shon AM, Ober C. Evidence of balancing selection at the HLA-G promoter region. Hum Mol Genet 2005;14:3619 –28. [14] Lajoie J, Jeanneau A, Faucher MC, Moreau P, Roger M. Characterisation of five novel HLA-G alleles with coding DNA base changes. Tissue Antigens 2008;72: 502– 4. [15] Mendes-Junior CT, Castelli EC, Simoes AL, Donadi EA. Absence of the HLAG*0105N allele in Amerindian populations from the Brazilian Amazon region: A possible role of natural selection. Tissue Antigens 2007;70:330 – 4. [16] Rahimi R, Zavaran Hosseini A, Yari F. Null allele frequencies at HLA-G locus in Iranian healthy subjects. Iran. J Immunol 2008;5:207–11. [17] Lin A, Li M, Xu DP, Zhang WG, Yan WH. Ethnic variation of the HLA-G*0105N allele in two Chinese populations. Tissue Antigens 2009;73:270 – 4. [18] Ober C, Aldrich C, Rosinsky B, Robertson A, Walker MA, Willadsen S, et al. HLA-G1 protein expression is not essential for fetal survival. Placenta 1998;19: 127–32. [19] Castro MJ, Morales P, Rojo-Amigo R, Martinez-Laso J, Allende L, Varela P, et al. Homozygous HLA-G*0105N healthy individuals indicate that membraneanchored HLA-G1 molecule is not necessary for survival. Tissue Antigens 2000;56:232–9. [20] Le Discorde M, Le Danff C, Moreau P, Rouas-Freiss N, Carosella ED. HLAG*0105N null allele encodes functional HLA-G isoforms. Biol Reprod 2005;73: 280 – 8. [21] Salzano FM, Callegari-Jacques SM. South American Indians: A Case Study in Evolution. Oxford: Oxford University Press; 1988. [22] Atay GA, Arsan S, Atasay B, Ensari A, Aysev D. The possible role of intrauterine infections in unexplained second trimester abortions and macerated stillbirths: A study from a single centre. J Perinatol 2004;24:679 – 85. [23] Petersson K, Norbeck O, Westgren M, Broliden K. Detection of parvovirus B19, cytomegalovirus and enterovirus infections in cases of intrauterine fetal death. J Perinat Med 2004;32:516 –21. [24] Salzano FM, Jacques SM. Genetic demography of the Central Pano and Kanamari Indians of Brazil. Hum Biol 1979;51:551– 64. [25] Salzano FM, Jacques SMC, Neel JV. Genetic demography of the Amazonian Ticuna Indians. J Hum Evol 1980;9:179 –91. [26] Mohrenweiser H, Neel JV, Mestriner MA, Salzano FM, Migliazza E, Simoes AL, et al. Electrophoretic variants in three Amerindian tribes: The Baniwa, Kanamari, and Central Pano of western Brazil. Am J Phys Anthropol 1979;50:237– 46. [27] Neel JV, Gershowitz H, Mohrenweiser HW, Amos B, Kostyu DD, Salzano FM, et al. Genetic studies on the Ticuna, an enigmatic tribe of Central Amazonas. Ann Hum Genet 1980;44:37–54. [28] Mendes-Junior CT, Simoes AL. Mitochondrial DNA variability among eight Tikuna villages: Evidence for an intratribal genetic heterogeneity pattern. Am J Phys Anthropol 2009;140:526 –31. [29] Luizon MR, Mendes-Junior CT, De Oliveira SF, Simoes AL. Ancestry informative markers in Amerindians from Brazilian Amazon. Am J Hum Biol 2008;20:86 –90.
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[30] Castelli EC, Mendes-Junior CT, Viana de Camargo JL, Donadi EA. HLA-G polymorphism and transitional cell carcinoma of the bladder in a Brazilian population. Tissue Antigens 2008;72:149 –57. [31] Parra FC, Amado RC, Lambertucci JR, Rocha J, Antunes CM, Pena SD. Color and genomic ancestry in Brazilians. Proc Natl Acad Sci USA 2003;100:177– 82. [32] Castelli EC, Mendes-Junior CT, Wiezel CE, Peres NT, Simoes AL, Rossi NM, et al. A novel HLA-G allele, HLA-G*010111, in the Brazilian population. Tissue Antigens 2007;70:349 –50.
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[33] Harris EE, Meyer D. The molecular signature of selection underlying human adaptations. Am J Phys Anthropol 2006;(suppl 43):89 –130. [34] Andres AM, Hubisz MJ, Indap A, Torgerson DG, Degenhardt JD, Boyko AR, et al. Targets of balancing selection in the human genome. Mol Biol Evol 2009;26: 2755– 64. [35] Ober C, Billstrand C, Kuldanek S, Tan Z. The miscarriage-associated HLA-G -725G allele influences transcription rates in JEG-3 cells. Hum Reprod 2006; 21:1743– 8.
Contents lists available at ScienceDirect
Absence of the HLA-G*0113N allele in Amerindian populations from the Brazilian Amazon region Celso T. Mendes-Junior a,*, Erick C. Castelli b, Philippe Moreau c, Aguinaldo L. SimÖes d, Eduardo A. Donadi b a
Departamento de Quìmica, Faculdade de Filosofia, Ciéncias e Letras de Ribeirâo Preto, Universidade de Sâo Paulo, 14040-901, Ribeirâo Preto-SP, Brazil Divisâo de Imunologia Clìnica, Departamento de Clìnica Mèdica, Universidade de Sâo Paulo, 14048-900, Ribeirâo Preto-SP, Brazil c Commissariat Þ l’Energie Atomique/DSV/I2BM/Service de Recherches en Hèmato-Immunologie, IUH, Hópital Saint-Louis, 75010 Paris, France d Departamento de Genètica, Faculdade de Medicina de Ribeirâo Preto, Universidade de Sâo Paulo, 14049-900, Ribeirâo Preto-SP, Brazil b
A R T I C L E
I N F O
Article history: Received 13 August 2009 Accepted 13 January 2010 Available online 4 February 2010
Keywords: Amerindians HLA-G Natural selection Null allele South America
A B S T R A C T
The HLA-G gene is predominantly expressed at the maternal-fetal interface and has been associated with maternal-fetal tolerance. The HLA-G*0113N is a null allele defined by the insertion of a premature stop codon at exon 2, observed in a single Ghanaian individual. Likewise the G*0105N allele, the occurrence of the HLA-G*0113N in a population from an area with high pathogen load suggests that the reduced HLA-G expression in G*0113N heterozygous placentas could improve the intrauterine defense against infections. The presence of the G*0113N allele here was investigated in 150 Amerindians from five isolated tribes that inhabit the Central Amazon and in 295 admixed individuals from the State of SÄo Paulo, Southeastern Brazil, previously genotyped for HLA-G. No copy of the G*0113N null allele was found in both population samples by exon 2 sequence-based analysis, reinforcing its restricted occurrence in Africa. 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.
1. Introduction The human leukocyte antigen G (HLA-G) gene is predominantly expressed at the maternal-fetal interface and has primarily been associated with maternal-fetal tolerance, enabling fetuses to survive unharmed in a genetically foreign host during successful pregnancies [1–3]. HLA-G is involved on the inhibition of cytotoxic T lymphocyte and natural killer cytolytic functions, preventing the proliferation of CD4⫹ T cells, and tolerizing dendritic cells through interactions with specific killer-cell immunoglobulin-like receptors and immunoglobulin-like transcript receptors expressed on these cells. Because of these interactions, HLA-G has been well recognized as a tolerogenic molecule [1]. The expression of HLA-G is not usually detected in normal tissues. In nonpathological conditions, HLA-G has been detected in the placenta, thymus, cornea, hematopoietic cells, proximal nail matrix, and pancreas. In pathologic situations, HLA-G expression has been observed in several tumors, viral infections, autoimmune diseases, and grafted tissues [4]. The HLA-G locus may generate multiple protein isoforms by alternative splicing of a single mRNA, giving rise to four membranebound (HLA-G1 to ⫺G4) and three soluble isoforms (HLA-G5 to ⫺G7) [1]. Compared with classical class I HLA loci, HLA-G exhibits a reduced coding-region genetic diversity, as has been observed in * Corresponding author. E-mail address: [email protected] (C.T. Mendes-Junior).
different populations [5–9]. Such lower degree of polymorphism of HLA-G may ensure the survival of the allogeneic fetus [2]. In fact, signatures of natural selection on the promoter and coding regions of the HLA-G gene, as well as in its vicinities, have been reported in worldwide populations [10 –13]. To date, 45 HLA-G alleles have been officially recognized, presenting sequence variations in coding and noncoding regions (IMGT ⫺ International Immunogenetics Information System, November 2009, http://www.ebi.ac.uk/imgt/ hla/stats.html). HLA-G alleles have been assigned to 17 distinct allele groups, generating 15 different membrane-bound proteins and two truncated nonfunctional HLA-G1 protein encoded by two null alleles. The first one, G*0105N, is defined by a ⌬C deletion at the last base of codon 129 or the first base of codon 130 in exon 3 that causes a shift in the reading frame [8,9]. The G*0105N allele is associated with an incomplete formation of the HLA-G1, ⫺G4, and ⫺G5 isoforms, and the expression of normal ⫺G2, ⫺G3, and ⫺G7 isoforms [1,9]. The second one, G*0113N [14], is defined by a C ¡ T transition at the first base of codon 54 (Fig. 1) in exon 2 (alpha-1 domain) that determines the presence of a premature TAG stop codon; such allele determines an incomplete formation of all HLA-G membrane-bound and soluble isoforms, resulting in presumably nonfunctional proteins [14]. Although the G*0105N allele frequencies vary across different geographic regions, being absent in Amerindians from the Brazilian Amazon, and ranging from 0% to 6.1% (Spain) in Europe, from 0% to 20.0% (Iran) in Asia, and from 4.8% (Ghana) to 11.1% (Zimbabwe) in
0198-8859/10/$32.00 - see front matter 䉷 2010 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved. doi:10.1016/j.humimm.2010.01.014
C.T. Mendes-Junior et al. / Human Immunology 71 (2010) 428 – 431
429
ity decline, which is happening even in the least developed countries, in which the average total fertility rate is now 5.2 children per woman. However, demographic data from six of the villages (from Katukina, Kaxinàwa, and Tikùna tribes) considered in the present study present a surprising average of 8.0 children per woman that reached the end of the reproductive period (ranging from 6.7 ⫾ 0.6 to 10.0 ⫾ 1.5) [24,25]. This suggests that the South-American Indians present unique cultural, environmental, demographic, and genetic features that render them special interest regarding the implications of HLA-G polymorphisms on fertility. 2. Subjects and methods
Fig. 1. Alignment of HLA-G alleles determined by nucleotide substitutions at exon 2 of such gene (complete sequence alignment of 44 HLA-G alleles identified by November 2009 – database release 2.27.1–is available at the Anthony Nolan Research Institute homepage, http://hla.alleles.org/data/hla-g.html).
sub-Saharan African populations (reviewed by [15–17]), the recently identified G*0113N was found in a single individual in a Ghanaian cohort of 100 samples [14]. Given the putative role of HLA-G in placental development, it has been proposed that the G*0105N allele is maintained in high frequency in some populations by non-neutral evolutionary forces [10]. The highest frequencies of G*0105N in geographic areas with a historically high pathogen load suggest that intrauterine pathogens may act as selective agents, increasing survival of heterozygous fetuses carrying the G*0105N allele in infected pregnancies (balancing selection). In this case, the reduced HLA-G1 expression in G*0105N heterozygous placentas may result in an overall increase in the number of T cells available in the uterus and reverse the inhibition of NK cells, improving the intrauterine defense against infections [10,18]. The survival of healthy individuals homozygous for the G*0105N allele [7,18,19] that do not express the G1, G4, and G5 soluble isoforms suggest that such isoforms are not essential for fetal survival in uncomplicated pregnancies [10,20]. Given that G*0113N transcripts do not result in any functional protein isoforms, the observation of a healthy homozygous individual for the G*0113N allele would be unexpected. Despite the different patterns of expression and functional consequences of G*0113N and G*0105N alleles, the evolutionary hypothesis proposed [10] for G*0105N might be suitable for G*0113N. However, the fact that the G*0113N allele was so far observed in a single Ghanaian individual [14] suggests a new mutational event that occurred in that individual or his/her family (private allele), keeping itself apart from the action of natural selection. By contrast, it is also possible that previous researches failed to identify such allele in worldwide populations since it was unknown. To further explore the population dynamics of G*0113N null allele, we investigated its occurrence among South American indigenous populations, in whom pregnancies have been rarely monitorized by specialized medical staff. In South America, particularly in the Amazon Rainforest, there are many pathogens [21] capable of triggering infections that may cause abortions and stillbirths [22,23]. Such intrauterine infections could have stronger effects on fetuses homozygous for functionally normal alleles than on heterozygous individuals carrying null alleles. Amerindian isolated populations exhibit high birth rates. According to a report prepared by the Department of Economic and Social Affairs of the United Nations (World Population Aging: 1950 – 2050, available at http://www.un.org/esa/population/publications/ worldageing19502050/), there is a worldwide trend toward fertil-
The population samples analyzed here were obtained from indigenous Brazilian populations visited in 1976 during the Alpha Helix expedition [26,27]. The 150 chosen Amerindians, without direct kinship, belonged to ten villages pertaining to five tribes (Katukina, Kaxinàwa, Marùbo, Tikùna, and Yaminawa) that inhabit the Central Amazon. Admixture levels with non-Amerindians were estimated to be definitely lower than 5% [26 –28]. Detailed descriptions of these populations, such as geographic distribution, linguistics, and demographic information, can be found elsewhere [26 –28]. Blood samples were processed and DNA was extracted as described elsewhere [12,15,29]. The G*0113N allele is defined by a C ¡ T transition at the first base of codon 54 in exon 2 that determines the presence of a premature TAG stop codon. The presence of such deletion was investigated according to a previously described method [6,30]: exon 2 was individually amplified yielding polymerase chain reaction (PCR) products of 361 bp, which were directly sequenced. The obtained sequences were aligned with the genomic sequences of the official alleles (recognized by the IMGT), and the existence of the G*0113N allele was carefully verified. Additionally, sequence data from 295 admixed individuals from the State of SÄo Paulo, Southeastern Brazil, previously genotyped for HLA-G exon two [6,30], were reexamined for the G*0113N mutation. 3. Results and discussion Since the first observations that HLA-G variability may have been shaped by non-neutral evolutionary forces, particularly balancing selection [10 –13], we have been studying HLA-G polymorphisms in Amerindian populations from the Brazilian Amazon [12,15] to evaluate and obtain some knowledge regarding such process in isolated South American populations. In a first attempt, the analysis of the 14-bp insertion/deletion polymorphism at the HLA-G 3= untranslated region (3= UTR) revealed a relatively low insertion frequency (38.54%) in 384 Amerindians and a trend toward balancing selection at this locus [12]. In a second effort, no copy of the G*0105N null allele was found in 143 Amerindians; such absence may be due to demographic or selective factors apart from balancing selection [15]. In the present study, the G*0113N null allele was not found in the Amerindian sample of 150 individuals (300 alleles). One may infer that the frequency of this allele is not higher than 1% in the Amerindian population, since otherwise, according to the binomial distribution, the probability of such finding (i.e., not observing the given allele in a sample of 300 alleles given that it exists in the population that originated the sampled individuals) would be lower than 5%. The lack of the null allele in the Amazonian Indians may be due to its probable absence in the migratory waves that originated from the gene pool of South American Indians. The support for this conclusion was derived from the fact that the G*0113N allele was observed in a single African individual so far [14]. Another possibility is that natural selection could have acted against HLA-G null alleles (G*0105N and G*0113N) in South American
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Indians, suggesting that membrane-bound isoforms are necessary for fetal defense against pathogens in the Amazonian environment. To further explore the existence of the G*0113N allele in South America, the reexamination of HLA-G exon 2 sequence data [6,30] from 295 admixed individuals (590 alleles) sampled from different places in the State of SÄo Paulo, Southeastern Brazil, confirmed the absence of such null allele in a highly diverse population. Following the same rationale described earlier in the text, it may be inferred that the frequency of the G*0113N allele is not higher than 0.5% in the Brazilian urban population. It should be emphasized that Brazilians represent one of the most heterogeneous populations in the world, which is the result of five centuries of genetic admixture between people from three continents: the European colonizers mainly represented by the Portuguese, the African slaves, and the autochthonous Amerindians [31]. Therefore, the Brazilian urban population has presented the largest HLA-G variability already detected [6,30,32]. Therefore, the present observation suggests that G*0113N is a Ghanaian private mutation rather than a worldwide polymorphic allele. Population genetics theory states that evolution is a dynamic process. Sometimes selection can vary over time, with different alleles being favored at different time intervals, as selective regimes change [33]. The great majority of random mutations that arise are neutral or deleterious. However, advantageous mutations can also arise by chance, allowing a higher fitness for a heterozygous individual compared with a homozygote. Much effort has been devoted to evaluate the action of balancing selection in the human genome [34]. Throughout evolutionary history, HLA-G expression levels may have evolved resulting in a fine balance between optimal levels of expression from fetal to adult life [13], allowing both fetal tolerance and appropriate intrauterine defense against pathogens: low HLA-G expression could stimulate recurrent abortions, whereas high HLA-G expression could provide susceptibility to intrauterine infection by pathogens. A high HLA-G expression would be acceptable in many geographic regions, but in areas where many intrauterine pathogens exist, both tolerance and defense would be desired. According to this hypothesis [10,13], in such specific environments a new mutation that would disrupt HLA-G expression could be advantageous in heterozygosis, providing intermediate HLA-G levels. Signatures of balancing selection on the promoter [13], coding [10], and 3= UTR [12] regions of the HLA-G gene, as well as the observation that variation in the HLA-G promoter region influences transcription rates and that increased expression of HLA-G may be deleterious in some pregnancies [35], support the heterozygous advantage hypothesis. It is possible that in pregnancies with subclinical infection, increased levels of HLA-G may compromise maternal cellular immunity to invading pathogens [35]. In conclusion, the G*0113N allele has not been observed in the autochtonous or admixed South-American populations considered herein. However, its absence in Asia and Europe, as well as its limited occurrence in Africa, should be confirmed by the reexamination of previously generated HLA-G exon 2 sequence data or by the evaluation of individuals from other population samples from such geographic areas. This would contribute to elucidate the geographic distribution and population dynamics of the G*0113N allele and to further explore the role of natural selection over the HLA-G gene. Acknowledgments The authors thank Dr. Simone Kashima, Sandra Rodrigues, Rochele Azevedo, Evandra Strazza Rodrigues and Adriana Marques for invaluable technical help. This study was supported by CNPq (Conselho Nacional de Desenvolvimento CientÎfico e TecnolÔgico - Brazil) Grant 475670/2007-08, CAPES (CoordenaÈÄo de AperfeiÈoamento de Pessoal de NÎvel Superior), FAPESP (FundaÈÄo de Amparo Á Pesquisa do Estado de SÄo Paulo) and FAEPA (FundaÈÄo de Apoio ao Ensino, Pesquisa e AssistËncia do HCFMRP-USP). C.T.M.J. was sup-
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