Study of the CCR5-m303 mutation in three different ethnic groups from Brazil
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《遗传学和分子生物学》
IFundao Instituto Oswaldo Cruz, Centro de Pesquisas Gonalo Moniz, Laboratorio Avanado de Saude Publica, Salvador, Ba, Brazil
IIFundao Bahiana para o Desenvolvimento das Ciências, Escola Bahiana de Medicina e Saude Publica, Salvador, Ba, Brazil
IIIUniversidade Federal da Bahia, Faculdade de Medicina, Departamento de Pediatria, Salvador, Ba, Brazil
IVUniversidade Federal da Bahia, Faculdade de Medicina, Departamento de Virologia, Salvador, Ba, Brazil
ABSTRACT
The main coreceptor gene involved in HIV-1 infection is CCR5 b chemokine receptor gene for which several mutations have been described, some of which have correlated with HIV-1 infection, acquired immune deficiency syndrome (AIDS), or both. Deletion of 32bp in the CCR5 gene (D32) has been shown to confer resistance to infection by HIV-1 R5 strains. Another mutation, characterized by a thymine to adenine (T to A) nucleotide substitution at position 303 (m303), has shown the same effects as the D32 mutation, with previous studies having shown that the allele frequency of the CCR5-m303 mutation is 0.014 in African-American and 0.007 in French populations. The Brazilian population is known to be genetically diverse, because of which we investigated the allele frequency of the CCR5-m303 mutation in three different Brazilian ethnic groups containing individuals who were not infected with HIV-1 and also in a cohort of HIV-1 long-term non-progressors. We used the polymerase chain reaction (PCR) and HincII restriction fragment length polymorphisms (RFLP) to investigate these populations and found that none of the 566 individuals examined the mutant CCR5-m303 allele. These results are in accordance with the previously reported allelic frequencies for African-American and Caucasian populations and may reflect the real prevalence of the m303 mutation in Brazil.
Key words: CCR5 gene, m303 frequency, Brazilian populations, HIV-1.
The b chemokine receptor CCR5, a member of the seven trans-membrane G-protein-coupled receptor family (Berger et al.1999), has been identified as a main coreceptor for entry of R5-tropic HIV-1 strains into target cells (e.g. CD4+ T- cells, monocytes/macrophages) (Alkhatib et al. 1996; Choe et al. 1996; Deng et al. 1996; Dragic et al. 1996; Doranz et al. 1996). The majority of HIV-1 variants occurring during the asymptomatic infection period are CCR5 tropic and are more frequently associated with in vivo transmission (Zhu et al. 1993). In contrast, the CXCR4 a-chemokine receptor is the coreceptor for the X4-tropic variants often found in the late period of infection (Feng et al. 1996).
Several studies have shown that mutations in the CCR5 gene affect both the HIV-1 infection process and the progression to AIDS. A 32 bp deletion (D32) in the CCR5 coding region has been shown to confer resistance to infection by R5 strains of HIV-1 (Dean et al. 1996; Liu et al. 1996). Moreover, as compared with peripheral blood mononuclear cells (PBMCs) from CCR5/CCR5 (wild-type) homozygotes, PBMCs from CCR5/D32 heterozygotes are less susceptible to in vitro infection by HIV-1 R5 strains (Deng et al. 1996; Berger et al. 1999; Grimaldi et al.2002), with seropositive CCR5/D32 individuals having shown delayed progression to AIDS (Dean et al. 1996; Huang et al. 1996).
Another mutation in the CCR5 gene is the m303 mutation, characterized by an open reading frame single T to A base pair transversion at nucleotide 303 which indicates a cysteine to stop codon change in the first extracellular loop of the b-chemokine receptor protein at amino acid 101 (C101X) (Carrington et al. 1997). The m303 mutation appears to play the same role as the D32 deletion while mutagenesis assays have not detected the expression of the m303 coreceptor on the surface of CCR5 null transfected cells which were found to be non-susceptible to HIV-1 R5-isolates in infection assays (Blanpain et al. 2000).
The m303 mutation has only been detected in heterozygosis in 2.86% (1/35) of African-American and 5.56% (1/18) of French (who also possessed the D32 allele in compound trans-heterozygosis) individuals at high risk for HIV-1 infection (Carrington et al. 1997; Quillent et al. 1998), indeed, Quillent et al. (1998) found as few as 1.4% of blood donors of undefined ethnic origin possessed the m303 mutation
Ometto et al. (1999) observed an m303 allelic frequency of 0.002 for uninfected Italian infants born to HIV-1 seropositive mothers and 0 for HIV-infected Italian children. The mutant m303 allele was not identified in 200 Kuwaitis (Voevodin et al. 1999), nor among 687 South Africans (Williamson et al. 2000) or in seronegative but HIV-1 infected Chinese (Shieh et al.1999; Wang et al. 2003), although the m303 gene was detected at an allelic frequency of 0.007 in 145 Caucasians (Williamson et al. 2000).
Since there is a great deal of genetic diversity in Brazilian human populations but a lack of reports concerning the m303 allele, we investigated the frequency of this allele in three different Brazilian seronegative ethnic groups and in a cohort of HIV-1 long-term non-progressors.
One seronegative group was from the city of Salvador, the capital of the northeastern Brazilian state of Bahia, where 80% of the 2.5 million inhabitants (Anon, 2000) are Afro-Brazilians or of mixed African and Portuguese (mestizo) descent (Azevedo et al. 1982). To form this group we randomly selected 400 people from a cross-sectional survey population from sentinel surveillance areas previously established for the investigation of various infectious diseases (Teixeira et al. 2002).
Another seronegative group was from the city of Joinville, the capital of the southern Brazilian state of Santa Catarina, the majority of the approximately 430,000 of this city being of German descendent (Anon, 2000). This group was composed of 50 blood-donors of German ancestry who had no reported risk behavior for sexually transmitted diseases.
A further seronegative group consisted of 50 individuals from each of two Amerindian tribes (the Tiriyo and the Waiampi) located in the northern Amazon river basin. The Tiriyo tribe speaks the Caribe language and inhabits a reservation on the Suriname-Brazil frontier, 750 of its 1,700 members living in Brazil, while the Waiampi tribe speaks Tupi-Guarani and lives on a reservation on the French Guiana-Brazil frontier with 450 of its 1,200 members living in the Brazilian state of Amapa. All the Amerindians (total = 100) included in the survey were HIV-1 negative.
The seropositive group consisted of 16 HIV-1 infected long-term non-progressor (LTNP) individuals selected from an outpatient clinic at the Federal University Hospital in Salvador, the inclusion criteria being 10 years or more of asymptomatic infection with no use of anti-retroviral therapy and CD4 counts above 500 cells/mm3.
Blood samples, collected with the informed consent of the participants, were obtained from the seronegative Amerindian groups between April and May 1997 and from the seronegative Salvador and seropositive HIV-1 groups between April and August 1998, the Joinville seronegative group being sampled between November 2000 and January 2001. Each individual provided 10 ml of blood, which was collected as described by Grimaldi et al. (2002) using ethylene-diamine tetra acetic acid (EDTA) as an anticoagulant. The blood plasma from each of the participants was screened for HIV types 1 and 2 using an enzyme-linked immunosorbent assay (ELISA) (Enzygnost Anti HIV-1/2 Plus-Behring, Marburg, Germany). Repeatedly reactive samples were submitted to Western Blotting (HIV Blot 2.2, Genelab Diagnostics, Singapore Science Park, Singapore) and were interpreted according to the manufacturer's instructions.
We extracted DNA from PBMCs and whole blood using a commercial kit (DNAzol, GIBCO-BRL, Rockville, USA). The CCR5-m303 gene was amplified by the PCR method described by Carrington et al. (1997) using 100 ng of sample DNA and a Perkin-Elmer 9600 thermal cycler (Perkin-Elmer, Connecticut, USA). The m303 mutation generates a premature stop codon in the CCR5 gene which deactivates the HincII restriction site, which means that the HincII restriction enzyme can be used to detect the m303 mutation. We used the Hincll 5U restriction enzyme (BioLabs Inc., New England, USA.) to digest the PCR products in a final volume of 10 mL and an incubation period of three hours at 37 °C. The RFLP products were separated by electrophoresis on 1.5% agarose gel and visualized by standard techniques. The CCR5/CCR5 wild-type genotype was detected by 240 and 46 bp bands while CCR5/m303 was represented by 286, 240 and 46 bp; and m303/m303 by a single band of 286 bp.
We did not detect the m303 allele in any of the 566 individuals sampled, showing that his mutation was absent from the four groups studied (Figure 1).
It has been suggested (Winkler et al., 2004) that infectious diseases can exert a selective advantage on some mutations, and it may be that the m303 allele is currently being subjected to such a process and spreading through populations of different ethnic origin as a result of its influence on HIV-1 infection and progression to AIDS. In our study there was no evidence of the presence of the m303 allele in any of the groups, including the HIV-1 LTNP group, suggesting that this event has not reached detectable levels yet.
Our results for the Salvador seronegative group (80% of African-Brazilian or mestizo origin are in agreement with those published by Carrington et al. (1997) for a cohort of highly exposed uninfected individuals and by Williamson et al. (2000) who found no evidence of the m303 allele in individuals of African origin. Although previous reports have detected the m303 allele in Caucasians (Quillent et al. 1998; Williamson et al. 2000) we did not detect it in the Joinville seronegative group, which consisted of blood donors of German descent. This could be because the sample-size (50 individuals) was small or because of miscegenation. Since previous studies (Voevodin et al. 1999; Shieh et al.1999; Wang et al. 2003) have not detected the m303 allele in Asian populations we did not expect to find it in either the Waiampi or Tiriyo populations because they are of Asian descent, and this was indeed the case.
In summary, our data indicates that the m303 allele is not prevalent in three HIV-1 seronegative Brazilian populations of different ethnic origin nor does it occur in a group of long-term non-progressors from Salvador.
Acknowledgement
This work was partially supported by the Fundao de Amparo a Pesquisa do Estado da Bahia (FAPESB), and by Coordenao de Aperfeioamento de Pessoal em Nivel Superior (CAPES).
References
Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM and Berger EA (1996) CC CKR5: A RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955-1958.
Anon (2000) Instituto Brasileiro de Geografia e estatistica (IBGE) http://www.ibge.gov.br.
Azevedo ES, Fortuna CMM and Silva KMC (1982) Spread and diversity of human population in Bahia, Brazil. Hum Biol 54:329-341.
Berger EA, Murphy PM and Farber J (1999) Chemokine receptors as HIV-1 coreceptors: Roles in viral entry, tropism, and disease. Annu Rev Immunol 17:657-700.
Blanpain C, Lee B, Tackoen M, Puffer B, Boom A, Libert F, Sharron M, Wittamer V, Vassart G, Doms RW and Parmentier M (2000) Multiple nonfunctional alleles of CCR5 are frequent in various human populations. Blood 96:1638-1645.
Carrington M, Kissner T, Gerrard B, Ivanov S, O'brien SJ and Dean M (1997) Novel alleles of the chemokine-receptor gene CCR5. Am J Hum Genet 61:1261-1267.
Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C and Sodroski J (1996) The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135-1148.
Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton RE, Hill CM, Davis CB, Peiper SC, Schall TJ, Littman DR and Landau NR (1996) Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661-666.
Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, Goedert JJ, Buchbinder SP, Vittinghoff E, Gomperts E, Donfield S, Vlahov D, Kaslow R, Saah A, Rinaldo C, Detels RSJ and O'Brien SJ (1996) Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 273:1856-1862.
Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, Parmentier M, Collman RG and Doms RW (1996) A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149-1158.
Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Maddon PJ, Koup RA, Moore JP and Paxton WA (1996) HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667-673.
Feng Y, Broder CC, Kennedy PE and Berger EA (1996) HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-877.
Grimaldi R, Shindo N, Acosta AX, Dourado I, Brites C, de Melo Carvalho O, Brito I, Bou-Habib DC and Galvao-Castro B (2002) Prevalence of the CCR5Delta32 mutation in Brazilian populations and cell susceptibility to HIV-1 infection. Hum Genet 111:102-104.
Huang Y, Paxton WA, Wolinsky SM, Neumann AU, Zhang L, He T, Kang S, Ceradini D, Jin Z, Yazdanbakhsh K, Kunstman K, Erickson D, Dragon E, Landau NR, Phair J, Ho DD and Koup RA (1996) The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nature Medicine 2:1240-1243.
Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup RA and Landau NR (1996) Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86:367-377.
Ometto L, Bertorelle R, Mainardi M, Giurisato M, Chieco-Bianchi L and De Rossi A (1999) Analysis of the CC chemokine receptor 5 m303 mutation in infants born to HIV-1-seropositive mothers. AIDS 13:871-872.
Quillent C, Oberlin E, Braun J, Rousset D, Gonzalez-Canali G, Metais P, Montagnier L, Virelizier JL, Arenzana-Seisdedos F and Beretta A (1998) HIV-1-resistance phenotype conferred by combination of two separate inherited mutations of CCR5 gene. Lancet 351:14-18.
Shieh B, Liau YE, Yan YP, Sun HS, Chen MY, Liu YC, Ko NY and Li C (1999) Alleles that may influence HIV-1 pathogenesis in Chinese subjects. AIDS 13:421-424.
Teixeira MG, Barreto ML and Costa MNC (2002) Sentinel areas: A monitoring strategy in public health. Cadernos de Saude Publica 18:1189-1195.
Voevodin A, Samilchuk E and Dashti S (1999) Frequencies of SDF-1 chemokine, CCR-5, and CCR-2 chemokine receptor gene alleles conferring resistance to human immunodeficiency virus type 1 and AIDS in Kuwaitis. J Med Virol 58:54-58.
Wang FS, Hong WG, Cao Y, Liu MX, Jin L, Hu LP, Wang Z, Feng TJ, Hou J, Zhang B, Shi M, Xu DP, Lei ZY, Wang B, Liu ZD, Ye JJ, Peng L, Qiu Y and Winkler C (2003) Population survey of CCR5 D32, CCR5 m303, CCR2b 64I, and SDF1 3'A allele frequencies in indigenous Chinese healthy individuals, and in HIV-1-infected and HIV-1-uninfected individuals in HIV-1 risk groups. J Acquir Immune Defic Syndr 32:124-130.
Williamson C, Loubser AS, Brice B, Joubert G, Smit T, Thomas R, Visagie M, Cooper M, Van Der Ryst E (2000). Allelic frequencies of host genetic variants influencing susceptibility to HIV-1 infection and disease in South African populations AIDS 14:449-451.
Winkler C, An P and O'Brien SJ (2004) Patterns of ethnic diversity among the genes that influence AIDS. Hum Mol Genet 1:13 Special number 1:R9-19.
Zhu T, Mo H, Wang N, Nam DS, Cao Y, Koup RA, Ho DD (1993) Genotypic and phenotypic characterization of HIV-1 in patients with primary infection. Science 261:1179-1181.(Rogerio Grimaldi, ; Angel)
IIFundao Bahiana para o Desenvolvimento das Ciências, Escola Bahiana de Medicina e Saude Publica, Salvador, Ba, Brazil
IIIUniversidade Federal da Bahia, Faculdade de Medicina, Departamento de Pediatria, Salvador, Ba, Brazil
IVUniversidade Federal da Bahia, Faculdade de Medicina, Departamento de Virologia, Salvador, Ba, Brazil
ABSTRACT
The main coreceptor gene involved in HIV-1 infection is CCR5 b chemokine receptor gene for which several mutations have been described, some of which have correlated with HIV-1 infection, acquired immune deficiency syndrome (AIDS), or both. Deletion of 32bp in the CCR5 gene (D32) has been shown to confer resistance to infection by HIV-1 R5 strains. Another mutation, characterized by a thymine to adenine (T to A) nucleotide substitution at position 303 (m303), has shown the same effects as the D32 mutation, with previous studies having shown that the allele frequency of the CCR5-m303 mutation is 0.014 in African-American and 0.007 in French populations. The Brazilian population is known to be genetically diverse, because of which we investigated the allele frequency of the CCR5-m303 mutation in three different Brazilian ethnic groups containing individuals who were not infected with HIV-1 and also in a cohort of HIV-1 long-term non-progressors. We used the polymerase chain reaction (PCR) and HincII restriction fragment length polymorphisms (RFLP) to investigate these populations and found that none of the 566 individuals examined the mutant CCR5-m303 allele. These results are in accordance with the previously reported allelic frequencies for African-American and Caucasian populations and may reflect the real prevalence of the m303 mutation in Brazil.
Key words: CCR5 gene, m303 frequency, Brazilian populations, HIV-1.
The b chemokine receptor CCR5, a member of the seven trans-membrane G-protein-coupled receptor family (Berger et al.1999), has been identified as a main coreceptor for entry of R5-tropic HIV-1 strains into target cells (e.g. CD4+ T- cells, monocytes/macrophages) (Alkhatib et al. 1996; Choe et al. 1996; Deng et al. 1996; Dragic et al. 1996; Doranz et al. 1996). The majority of HIV-1 variants occurring during the asymptomatic infection period are CCR5 tropic and are more frequently associated with in vivo transmission (Zhu et al. 1993). In contrast, the CXCR4 a-chemokine receptor is the coreceptor for the X4-tropic variants often found in the late period of infection (Feng et al. 1996).
Several studies have shown that mutations in the CCR5 gene affect both the HIV-1 infection process and the progression to AIDS. A 32 bp deletion (D32) in the CCR5 coding region has been shown to confer resistance to infection by R5 strains of HIV-1 (Dean et al. 1996; Liu et al. 1996). Moreover, as compared with peripheral blood mononuclear cells (PBMCs) from CCR5/CCR5 (wild-type) homozygotes, PBMCs from CCR5/D32 heterozygotes are less susceptible to in vitro infection by HIV-1 R5 strains (Deng et al. 1996; Berger et al. 1999; Grimaldi et al.2002), with seropositive CCR5/D32 individuals having shown delayed progression to AIDS (Dean et al. 1996; Huang et al. 1996).
Another mutation in the CCR5 gene is the m303 mutation, characterized by an open reading frame single T to A base pair transversion at nucleotide 303 which indicates a cysteine to stop codon change in the first extracellular loop of the b-chemokine receptor protein at amino acid 101 (C101X) (Carrington et al. 1997). The m303 mutation appears to play the same role as the D32 deletion while mutagenesis assays have not detected the expression of the m303 coreceptor on the surface of CCR5 null transfected cells which were found to be non-susceptible to HIV-1 R5-isolates in infection assays (Blanpain et al. 2000).
The m303 mutation has only been detected in heterozygosis in 2.86% (1/35) of African-American and 5.56% (1/18) of French (who also possessed the D32 allele in compound trans-heterozygosis) individuals at high risk for HIV-1 infection (Carrington et al. 1997; Quillent et al. 1998), indeed, Quillent et al. (1998) found as few as 1.4% of blood donors of undefined ethnic origin possessed the m303 mutation
Ometto et al. (1999) observed an m303 allelic frequency of 0.002 for uninfected Italian infants born to HIV-1 seropositive mothers and 0 for HIV-infected Italian children. The mutant m303 allele was not identified in 200 Kuwaitis (Voevodin et al. 1999), nor among 687 South Africans (Williamson et al. 2000) or in seronegative but HIV-1 infected Chinese (Shieh et al.1999; Wang et al. 2003), although the m303 gene was detected at an allelic frequency of 0.007 in 145 Caucasians (Williamson et al. 2000).
Since there is a great deal of genetic diversity in Brazilian human populations but a lack of reports concerning the m303 allele, we investigated the frequency of this allele in three different Brazilian seronegative ethnic groups and in a cohort of HIV-1 long-term non-progressors.
One seronegative group was from the city of Salvador, the capital of the northeastern Brazilian state of Bahia, where 80% of the 2.5 million inhabitants (Anon, 2000) are Afro-Brazilians or of mixed African and Portuguese (mestizo) descent (Azevedo et al. 1982). To form this group we randomly selected 400 people from a cross-sectional survey population from sentinel surveillance areas previously established for the investigation of various infectious diseases (Teixeira et al. 2002).
Another seronegative group was from the city of Joinville, the capital of the southern Brazilian state of Santa Catarina, the majority of the approximately 430,000 of this city being of German descendent (Anon, 2000). This group was composed of 50 blood-donors of German ancestry who had no reported risk behavior for sexually transmitted diseases.
A further seronegative group consisted of 50 individuals from each of two Amerindian tribes (the Tiriyo and the Waiampi) located in the northern Amazon river basin. The Tiriyo tribe speaks the Caribe language and inhabits a reservation on the Suriname-Brazil frontier, 750 of its 1,700 members living in Brazil, while the Waiampi tribe speaks Tupi-Guarani and lives on a reservation on the French Guiana-Brazil frontier with 450 of its 1,200 members living in the Brazilian state of Amapa. All the Amerindians (total = 100) included in the survey were HIV-1 negative.
The seropositive group consisted of 16 HIV-1 infected long-term non-progressor (LTNP) individuals selected from an outpatient clinic at the Federal University Hospital in Salvador, the inclusion criteria being 10 years or more of asymptomatic infection with no use of anti-retroviral therapy and CD4 counts above 500 cells/mm3.
Blood samples, collected with the informed consent of the participants, were obtained from the seronegative Amerindian groups between April and May 1997 and from the seronegative Salvador and seropositive HIV-1 groups between April and August 1998, the Joinville seronegative group being sampled between November 2000 and January 2001. Each individual provided 10 ml of blood, which was collected as described by Grimaldi et al. (2002) using ethylene-diamine tetra acetic acid (EDTA) as an anticoagulant. The blood plasma from each of the participants was screened for HIV types 1 and 2 using an enzyme-linked immunosorbent assay (ELISA) (Enzygnost Anti HIV-1/2 Plus-Behring, Marburg, Germany). Repeatedly reactive samples were submitted to Western Blotting (HIV Blot 2.2, Genelab Diagnostics, Singapore Science Park, Singapore) and were interpreted according to the manufacturer's instructions.
We extracted DNA from PBMCs and whole blood using a commercial kit (DNAzol, GIBCO-BRL, Rockville, USA). The CCR5-m303 gene was amplified by the PCR method described by Carrington et al. (1997) using 100 ng of sample DNA and a Perkin-Elmer 9600 thermal cycler (Perkin-Elmer, Connecticut, USA). The m303 mutation generates a premature stop codon in the CCR5 gene which deactivates the HincII restriction site, which means that the HincII restriction enzyme can be used to detect the m303 mutation. We used the Hincll 5U restriction enzyme (BioLabs Inc., New England, USA.) to digest the PCR products in a final volume of 10 mL and an incubation period of three hours at 37 °C. The RFLP products were separated by electrophoresis on 1.5% agarose gel and visualized by standard techniques. The CCR5/CCR5 wild-type genotype was detected by 240 and 46 bp bands while CCR5/m303 was represented by 286, 240 and 46 bp; and m303/m303 by a single band of 286 bp.
We did not detect the m303 allele in any of the 566 individuals sampled, showing that his mutation was absent from the four groups studied (Figure 1).
It has been suggested (Winkler et al., 2004) that infectious diseases can exert a selective advantage on some mutations, and it may be that the m303 allele is currently being subjected to such a process and spreading through populations of different ethnic origin as a result of its influence on HIV-1 infection and progression to AIDS. In our study there was no evidence of the presence of the m303 allele in any of the groups, including the HIV-1 LTNP group, suggesting that this event has not reached detectable levels yet.
Our results for the Salvador seronegative group (80% of African-Brazilian or mestizo origin are in agreement with those published by Carrington et al. (1997) for a cohort of highly exposed uninfected individuals and by Williamson et al. (2000) who found no evidence of the m303 allele in individuals of African origin. Although previous reports have detected the m303 allele in Caucasians (Quillent et al. 1998; Williamson et al. 2000) we did not detect it in the Joinville seronegative group, which consisted of blood donors of German descent. This could be because the sample-size (50 individuals) was small or because of miscegenation. Since previous studies (Voevodin et al. 1999; Shieh et al.1999; Wang et al. 2003) have not detected the m303 allele in Asian populations we did not expect to find it in either the Waiampi or Tiriyo populations because they are of Asian descent, and this was indeed the case.
In summary, our data indicates that the m303 allele is not prevalent in three HIV-1 seronegative Brazilian populations of different ethnic origin nor does it occur in a group of long-term non-progressors from Salvador.
Acknowledgement
This work was partially supported by the Fundao de Amparo a Pesquisa do Estado da Bahia (FAPESB), and by Coordenao de Aperfeioamento de Pessoal em Nivel Superior (CAPES).
References
Alkhatib G, Combadiere C, Broder CC, Feng Y, Kennedy PE, Murphy PM and Berger EA (1996) CC CKR5: A RANTES, MIP-1alpha, MIP-1beta receptor as a fusion cofactor for macrophage-tropic HIV-1. Science 272:1955-1958.
Anon (2000) Instituto Brasileiro de Geografia e estatistica (IBGE) http://www.ibge.gov.br.
Azevedo ES, Fortuna CMM and Silva KMC (1982) Spread and diversity of human population in Bahia, Brazil. Hum Biol 54:329-341.
Berger EA, Murphy PM and Farber J (1999) Chemokine receptors as HIV-1 coreceptors: Roles in viral entry, tropism, and disease. Annu Rev Immunol 17:657-700.
Blanpain C, Lee B, Tackoen M, Puffer B, Boom A, Libert F, Sharron M, Wittamer V, Vassart G, Doms RW and Parmentier M (2000) Multiple nonfunctional alleles of CCR5 are frequent in various human populations. Blood 96:1638-1645.
Carrington M, Kissner T, Gerrard B, Ivanov S, O'brien SJ and Dean M (1997) Novel alleles of the chemokine-receptor gene CCR5. Am J Hum Genet 61:1261-1267.
Choe H, Farzan M, Sun Y, Sullivan N, Rollins B, Ponath PD, Wu L, Mackay CR, LaRosa G, Newman W, Gerard N, Gerard C and Sodroski J (1996) The beta-chemokine receptors CCR3 and CCR5 facilitate infection by primary HIV-1 isolates. Cell 85:1135-1148.
Deng H, Liu R, Ellmeier W, Choe S, Unutmaz D, Burkhart M, Di Marzio P, Marmon S, Sutton RE, Hill CM, Davis CB, Peiper SC, Schall TJ, Littman DR and Landau NR (1996) Identification of a major co-receptor for primary isolates of HIV-1. Nature 381:661-666.
Dean M, Carrington M, Winkler C, Huttley GA, Smith MW, Allikmets R, Goedert JJ, Buchbinder SP, Vittinghoff E, Gomperts E, Donfield S, Vlahov D, Kaslow R, Saah A, Rinaldo C, Detels RSJ and O'Brien SJ (1996) Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Science 273:1856-1862.
Doranz BJ, Rucker J, Yi Y, Smyth RJ, Samson M, Peiper SC, Parmentier M, Collman RG and Doms RW (1996) A dual-tropic primary HIV-1 isolate that uses fusin and the beta-chemokine receptors CKR-5, CKR-3, and CKR-2b as fusion cofactors. Cell 85:1149-1158.
Dragic T, Litwin V, Allaway GP, Martin SR, Huang Y, Nagashima KA, Cayanan C, Maddon PJ, Koup RA, Moore JP and Paxton WA (1996) HIV-1 entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature 381:667-673.
Feng Y, Broder CC, Kennedy PE and Berger EA (1996) HIV-1 entry cofactor: Functional cDNA cloning of a seven-transmembrane, G protein-coupled receptor. Science 272:872-877.
Grimaldi R, Shindo N, Acosta AX, Dourado I, Brites C, de Melo Carvalho O, Brito I, Bou-Habib DC and Galvao-Castro B (2002) Prevalence of the CCR5Delta32 mutation in Brazilian populations and cell susceptibility to HIV-1 infection. Hum Genet 111:102-104.
Huang Y, Paxton WA, Wolinsky SM, Neumann AU, Zhang L, He T, Kang S, Ceradini D, Jin Z, Yazdanbakhsh K, Kunstman K, Erickson D, Dragon E, Landau NR, Phair J, Ho DD and Koup RA (1996) The role of a mutant CCR5 allele in HIV-1 transmission and disease progression. Nature Medicine 2:1240-1243.
Liu R, Paxton WA, Choe S, Ceradini D, Martin SR, Horuk R, MacDonald ME, Stuhlmann H, Koup RA and Landau NR (1996) Homozygous defect in HIV-1 coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1 infection. Cell 86:367-377.
Ometto L, Bertorelle R, Mainardi M, Giurisato M, Chieco-Bianchi L and De Rossi A (1999) Analysis of the CC chemokine receptor 5 m303 mutation in infants born to HIV-1-seropositive mothers. AIDS 13:871-872.
Quillent C, Oberlin E, Braun J, Rousset D, Gonzalez-Canali G, Metais P, Montagnier L, Virelizier JL, Arenzana-Seisdedos F and Beretta A (1998) HIV-1-resistance phenotype conferred by combination of two separate inherited mutations of CCR5 gene. Lancet 351:14-18.
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