Recent publications have defined the role of chemokine receptor 5 (CCR5) as a coreceptor for HIV-1 infection(1-3). A 32-bp deletion in CCR5 (CCR5del32) confers resistance to HIV-1 infection and disease progression in men. The deletion causes a frame shift at amino acid 185, and the truncated protein is not expressed on the surface of the cell(4). One report shows that the frequency of CCR5del32 in infected men (0.054) is nearly half that of uninfected men (0.092), suggesting that the allele has a dominant protective effect for infection(5). Other reports indicate that no differences in allele frequencies exist between infected and uninfected adults(6,7). In addition, Dean et al.(6) found a higher frequency of heterozygosity for the CCR5del32 allele in HIV-1-infected long-term nonprogressors (31%) compared with rapid progressors (16%) among homosexual male but not hemophiliac risk groups, suggesting that a copy of CCR5del32 is protective for disease progression within the homosexual adult male risk group. Because the effect of CCR5del32 is different in these risk groups, it is possible that route of transmission or viral load may affect the level of protection that a single copy of the mutant gene can confer. Therefore, the next question to address is: What effect does CCR5 have in perinatal HIV-1 transmission and disease progression?
In children perinatally infected with HIV-1, expression of clinical signs of disease follows a bimodal distribution(8,9). In approximately 20% of cases, children have a rapid progression to immunodeficiency and death within the first 2 years of life, whereas the majority progress much more slowly, surviving to a median of 8 years of age(8,10). Multiple factors have been shown to affect HIV-1 disease progression in perinatally infected children. These factors include maternal disease status at time of delivery(8), therapeutic and prophylactic treatments of the mother during gestation and of the infant after birth(11,12), and host human leukocyte antigen(HLA) genotype(13). We have investigated the effect of CCR5del32 among perinatally exposed African American and Catalan children and found no significant association of CCR5del32 with HIV-1 infection and disease progression.
Participants were drawn from two cohorts: the first from Harlem Hospital Center (New York, NY, U.S.A.) and the second from five hospitals in and around Barcelona, Spain. Signed, informed consent was obtained from a parent or guardian for all children enrolled in this study. Institutional review board approval for the study was obtained from all participating institutions. Children from both cohorts were identified as HIV-1 infected by persistence of IgG antibodies to HIV-1 15 months after birth, positive results on two HIV-1 viral tests (culture, polymerase chain reaction [PCR], or presence of antigen), or AIDS-defining illness according to Centers for Disease Control and Prevention(CDC) criteria(14).
Cohort 1 consisted of 144 African American children from Harlem Hospital. Enrollment took place from December 1989 to March of 1994 and comprised all 51 children seen for prevalent HIV-1 infection by one of the authors (E.J.A.) and all 93 children born at Harlem Hospital to HIV-1 infected mothers for whom said author was the attending pediatrician. Among the 93 prospectively enrolled children, 42 were infected with HIV-1 and 51 were seroreverters. None of the prospectively enrolled children had mothers who used azidothymidine (AZT) during pregnancy, a drug that has been shown to significantly decrease perinatal HIV-1 transmission(11). All HIV-1-infected children, including both prevalent and prospective cases, had available information regarding clinical manifestations of disease. Because this set includes prevalent cases, it is biased toward older children and long-term survivors. Children were evaluated for rate of disease progression based on the presence or absence of the following clinical manifestations: incidence of encephalopathy, opportunistic infections, or death before 2 years of age.
Cohort 2 consisted of 73 HIV-1-infected children of Spanish or Basque ancestry identified at the five largest hospitals in and around Barcelona. Children were enrolled from January 1994 to December 1994 during routine clinic visits, which took place approximately every 3 months. Ages of the children ranged from 5 months to 9 years. A standardized questionnaire was used by the attending pediatrician to obtain clinical data. The CDC revised classification system for HIV in children <13 years of age was used to categorize disease progression status(15). The disease progression categories were based on either clinical signs and symptoms of disease or immunologic categories based on CD4 counts. Previous studies indicate a high degree of correlation between clinical and immunologic parameters in this cohort(16). Because cohort 2 represents prevalent cases of HIV-1 infection, this group is biased toward older children and long-term survivors.
DNA was extracted from blood by standard techniques(17). A PCR amplification followed by agarose gel electrophoresis was used to identify CCR5del32. The primers (i.e., SP4.760, PM6.942) were selected from a previous publication(4). Odds ratios were calculated and statistical significance assessed with a χ2 or Fisher's exact test.
CCR5del32 and Infection
The 93 prospectively enrolled children in cohort 1 were analyzed for association of CCR5del32 with incidence of HIV-1 infection. Four children were heterozygous for the allele and none of the children were homozygous(Table 1). Among the 42 infected children, three were heterozygous, and among the 51 seroreverters, one was heterozygous. If CCR5del32 were protective for risk of infection in perinatally infected children, the clinician would expect to observe a higher frequency of heterozygotes in uninfected than in infected children. Among these children, heterozygosity for the deletion is more frequent in infected children (p = 0.32), suggesting that the allele is not protective for risk of infection.
CCR5del32 and Disease Progression
Encephalopathy, opportunistic infections, and death before 2 years of age are the most severe clinical manifestations of HIV-1 disease progression in children. Information about these conditions was ascertained for 93 HIV-1-infected African Americans from cohort 1 (both prevalent and prospective cases). The frequency of encephalopathy, opportunistic infections, and death before 2 years of age among these children was 16%, 17%, and 17%, respectively. Among the 93 HIV-1 infected children, three (3.2%) were heterozygous for the CCR5del32 allele and none were homozygous. The allele frequency in this group is 0.016. None of the 28 children who had at least one of these severe manifestations had the CCR5del32 allele, whereas three of 65 (5%) without severe manifestations had the allele. This difference was not statistically significant.
The CCR5del32 allele is not associated with long-term survival of children from cohort 1; among the 20 children who survived 8 years or more, none had the CCR5del32 allele. In addition, none of the 26 children who were 28 months or older at enrollment (average age, 5.0 years) had the CCR5del32 allele.
The CDC revised classification(15) was used to define clinical categories (i.e., asymptomatic, mildly symptomatic, moderately symptomatic, and severely symptomatic) and degree of immunosuppression based on age-specific CD4 T-lymphocyte counts in 73 HIV-1 infected children from cohort 2. Six children were heterozygous for CCR5del32 (allele frequency = 0.041). Among the heterozygotes, three were severely symptomatic, two were moderately symptomatic, and one was asymptomatic (Table 2). Similarly, when children were classified based on degree of immunosuppression, the frequency of the CCR5del32 allele was slightly increased in those with increased immunosuppression(data not shown), suggesting that CCR5del32 is not protective for disease progression.
Previous studies have found CCR5del32 frequencies in HIV-1-infected Caucasians to be 0.054,(5,6) similar to the allele frequency found among infected Catalan children in this study (0.041). The frequency of CCR5del32 among HIV-1-infected African American children (0.016) is similar to the frequency reported for adult African Americans participating in AIDS cohorts(6). Because the allele is presumed to be of Caucasian origin(5) and African Americans represent a mixture of genetic populations, primarily African and Caucasian, the frequency of CCR5del32 in African Americans may reflect the dilution of the primarily Caucasian allele. Because the association of CCR5del32 with disease progression is of small magnitude and the allele is present in such low frequency, the attributable risk of CCR5del32 is quite small, particularly among African Americans and probably other non-Caucasian ethnic groups.
Our study in perinatally exposed children finds that CCR5del32 does not have a dominant effect on HIV-1 infection. Our conclusion agrees with the findings in studies of homosexual men(6,7). However, unlike studies in Caucasian homosexual men, no significant association exists with a single copy of CCR5del32 and disease progression among the perinatally infected African American or Catalan children in our study. Allelic heterogeneity among the different ethnic groups, differences between perinatal and homosexual routes of transmission, or the differences in sample size may explain the unique results in these perinatal populations. Perhaps the cells of the immune system during perinatal and early childhood development are sufficiently different from those of adults to cause a different effect of CCR5del32. So far, no other polymorphisms in CCR5 that disrupt receptor function have been identified. Further molecular analysis of other polymorphisms in this gene will provide a more complete understanding of the effect of CCR5 on HIV-1 infection and disease progression. In addition, Michael et al.(18) suggest that CCR5del32 has an increased protective effect in individuals infected with macrophage-tropic nonsyncytium-inducing strains of HIV-1 as compared with syncytium-inducing T-cell tropic strains. Thus, viral phenotype may also play an important role in determining the effect of CCR5del32.
Acknowledgments: We would like to thank Dr. Leslie Louie for her critical comments and assistance and Dr. Elizabeth Schubert for review of the manuscript. This work was supported by grant R01HD25792 from the National Institute of Health and predoctoral fellowship F31RR05062-02 to C. M. Rousseau.
Hospital collaborators: Claudia Fortuny, Hospital Sant Joan de Deu; Antonio Mur, Hospital del Mar; Josep Ma Gertrand, Hospital Vall d'Hebron; and Carlos Rodrigo, Hospital Can Ruti.
1. Alkhatib G, Combadiere C, Broder CC, et al. CC CKR5: a RANTES, MIP-1 alpha, MIP-1 beta receptor as a fusion cofactor for macrophage-tropic HIV-1
2. Deng H, Liu R, Ellmeier W, Choe S, et al. Identification of a major co-receptor for primary isolates of HIV-1
3. Dragic T, Litwin V, Allaway GP, et al. HIV-1
entry into CD4+ cells is mediated by the chemokine receptor CC-CKR-5. Nature
4. Liu R, Paxton WA, Choe S, et al. Homozygous defect in HIV-1
coreceptor accounts for resistance of some multiply-exposed individuals to HIV-1
5. Samson M, Libert F, Doranz BJ, et al. Resistance to HIV-1
infection in Caucasian individuals bearing mutant alleles of the CCR-5 chemokine receptor gene. Nature
6. Dean M, Carrington M, Winkler C, et al. Genetic restriction of HIV-1
infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science
7. Huang Y, Paxton W, Wolinsky S, et al. The role of a mutant CCR5
allele in HIV-1
transmission and disease progression. Nature Medicine
8. Blanche S, Mayaux MJ, Rouzioux C, et al. Relation of the course of HIV infection in children to the severity of the disease in their mothers at delivery. N Engl J Med
9. Frederick T, Mascola L, Eller A, O'Neil L, Byers B. Progression of human immunodeficiency virus disease among infants and children infected perinatally with human immunodeficiency virus or through neonatal blood transfusion. Los Angeles County Pediatric AIDS Consortium and the Los Angeles County-University of Southern California Medical Center and the University of Southern California School of Medicine. Pediatr Infect Dis J
10. Tovo PA, de Martino M, Gabiano C, et al. Prognostic factors and survival in children with perinatal HIV-1
infection. The Italian Register for HIV Infections in Children. Lancet
11. Newell ML, Gibb DM. A risk-benefit assessment of zidovudine in the prevention of perinatal
HIV transmission. Drug Safety
12. Ogino MT, Dankner WM, Spector SA. Development and significance of zidovudine resistance in children infected with human immunodeficiency virus. J Pediatr
13. Just JJ, Abrams E, Louie LG, et al. Influence of host genotype on progression to acquired immunodeficiency syndrome among children infected with human immunodeficiency virus type 1. J Pediatr
14. Control CfD. Classification system for human immunodeficiency virus (HIV) infection in children under 13 years of age. MMWR Morb Mortal Wkly Rep
15. Caldwell M, Oxtoby M, Simonds R, Lindergren M, Rogers M. 1994 revised classification system for human immunodeficiency virus infection in children less than 13 years of age. MMWR Morb Mortal Wkly Rep
16. Just JJ, Casabona J, Bertran J, et al. MHC class II alleles associated with clinical and immunological manifestations of HIV-1
infection among children in Catalonia, Spain. Tissue Antigens
17. Hall JM, Zuppan PJ, Anderson LA, Huey B, Carter C, King MC. Oncogenes and human breast cancer. Am J Hum Genet
18. Michael NL, Chang G, Louie LG, et al. The role of viral phenotype and CCR-5 gene defects in HIV-1
transmission and disease progression.Nature Medicine