INTRODUCTION

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Viral, immunologic, and genetic host factors are known to influence the risk of human immunodeficiency virus (HIV) infection and the rate of disease progression. Although the role of the CD4 molecule as a high-affinity receptor which is necessary but not sufficient for HIV entry has been known for a long time, it is only recently that the identity and contribution of other coreceptors in HIV infection and disease pathogenesis have begun to be recognized. One such receptor is the -chemokine receptor 5 (CCR5) to which macrophage-tropic (M-tropic) strains of HIV-1 must bind in order to enter the CD4 cell (1, 4, 8, 9).  Chemokines RANTES, MIP-1, MIP-1 are the natural ligands of CCR5 and inhibit the entry of M-tropic viruses into the cells (5). M-tropic non-syncytium-inducing viruses are important for establishing infection by the mucosal route and through inoculation of blood and are believed to play a role in mother-infant transmission of HIV (16). They are the predominant viruses early in the disease course. In contrast, viruses in advanced disease are predominantly T cell tropic, syncytium-inducing viruses (6, 14, 17) and utilize the other coreceptor, CXCR-4 (previously known as fusin or LESTR), which like CCR5 is a seven-transmembrane protein whose ligand has been identified as the stromal cell-derived factor (10).

A genetic mutation in the CCR5 gene consisting of 32-bp deletion (CCR532 [32]) results in a nonfunctional chemokine receptor. Homozygosity for this allele (32/32) confers strong resistance to infection by HIV (12, 13). The 32 allele has also been reported to influence the rate of disease progression, although the data is conflicting (7, 11).

The frequency of the 32 gene in the HIV-infected pediatric population and its influence on disease progression in HIV-infected children are relatively unknown. The objectives of this study were to assess the distribution of 32 in a population of HIV-infected children and to study the effect of the 32 mutation on the course of the disease. No child was found to be homozygous for 32. Two infected subjects, both non-rapid progressors, were heterozygous for 32 (wild type [wt]/32), but the 32 allele was not identified in any child with rapid disease progression. Although the difference in the prevalence of wt/32 in rapid disease progressors compared to non-rapid disease progressors did not achieve statistical significance (P = 0.076), analysis of disease severity in this cohort suggests that 32 heterozygosity may contribute to slowing disease progression.

	MATERIALS AND METHODS

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Patients. Forty-seven pediatric patients (43 HIV infected, 2 exposed to HIV [mothers were HIV positive], and 2 not exposed to HIV [mothers were HIV negative]) were evaluated. The infected children had acquired disease through the perinatal route (n = 41) or through infected blood products (2 hemophiliacs). Children in the perinatal-infection group ranged in age from 1.7 to 18.8 years. There were 24 males and 17 females. The two hemophiliacs were 16.6 and 19.5 years of age. Ethnically there were 63% African-Americans, 15% Caucasians, 10% Latinos, 5% of mixed parentage, 5% Asians, and 2% of unknown ethnic background.

CCR5 genotyping. (See reference 11.) DNA was isolated from stored peripheral blood mononuclear cells of 43 HIV infected, 2 uninfected HIV-exposed, and 2 uninfected unexposed children. To detect the presence or absence of the 32 allele, a PCR-based assay using primers flanking the 32-bp segment was used to amplify this region of the CCR5 gene. The presence or absence of the deletion was then determined by gel electrophoresis as follows.

	RESULTS

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None of the HIV-infected children had the homozygous gene for 32. The mutant CCR5 allele (wt/32) was present in 2 (4.2%) of the 47 childrenin none of the 7 rapid disease progressors; in 2 of the 17 non-rapid disease progressors (11.76%) and of the 36 children in the combined group of non-rapid progressors and those with intermediate disease course (5.5%) (Table 1). Differences in the prevalence of the mutant CCR5 gene between any of the groups were not significant by Fisher's exact test, although there was a trend towards significance in the prevalence of wt/32 in non-rapid progressors compared to the total study group (P = 0.076). There were no deaths on follow-up to average ages of 3.7 (1.7 to 5.7) years for the rapid progressors, 12.8 (8.4 to 19.4) years for non-rapid progressors, and 5.8 (3 to 7.9) years for the intermediate group. None of the uninfected HIV-exposed or uninfected unexposed children had the mutant allele. The frequency of the mutant CCR5 allele in this multiethnic population (African-Americans, Caucasians, Latinos, and Asians) is ~4.8%.

Clinical details of patients with 32 heterozygosity are described below.

Case 1. The first patient was an 8-year-old boy born to an HIV-positive African-American mother and an unknown father. He had fetal alcohol syndrome and thalassemia minor. The child was first diagnosed as having HIV infection at 3 years of age when he had septic arthritis and meningitis (Haemophilus influenza type B), which led to bilateral severe sensorineural hearing loss, mild microcephaly, and decreased visual motor coordination. At the time of the study, he had severe immunosuppression (CD4⁺, 1% [absolute count, 13]; CD4/CD8 ratio, 0.03) and viral load of 49,370 RNA copies (4.69 log₁₀)/ml.

Case 2. The second patient was a 19 1/2-year-old Caucasian male with hemophilia A infected by contaminated blood products prior to March 1985. He was diagnosed as having HIV infection in 1988 and had received antiretroviral monotherapy in the past. His clinical course had been marked by drug reactions, episodes of pancreatitis, chronic herpes zoster, and spontaneous pneumothorax requiring blebectomy. At the time of the study, he had severe immunosuppression (no detectable CD4⁺ cells for 2 years) and a viral load of >750,000 RNA copies/ml.

	DISCUSSION

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The importance of chemokine receptors for HIV entry and disease pathogenesis is becoming increasingly apparent (7). A genetic defect consisting of a deletion of 32 bp in the coding sequence from position 794 to position 825 (32) has been associated with resistance to HIV infection (13). Our study was undertaken to evaluate the frequency of 32 in children with HIV infection and to study its influence on disease pathogenesis.

Two patients heterozygous for 32 were identified among HIV-positive subjects in this study. In agreement with previous studies, these findings indicate that heterozygosity for 32 does not protect individuals from HIV infection. Several large studies have shown that the frequencies of wt/32 in HIV-infected and HIV-negative individuals are similar (7).

The frequency of the heterozygous 32 in our study of HIV-exposed and -infected children predominantly of nonwhite background was 4.8%. This frequency is similar to the 1.7% frequency reported by Dean et al. for the African American population (7). Studies in large populations show that the mutant CCR5 gene is variably distributed in different racial groups. In the Western European Caucasian populations, the 32 allele is found at the high frequency of ~0.20 but has not been found in any individuals of West African or Central African origin, in the Japanese, or in other ethnic groups (13).

None of the 43 HIV-infected children studied by us was homozygous for the 32 gene. The presence of two 32 alleles was initially described for two individuals who had remained uninfected despite repeated exposure to HIV (12). Peripheral blood mononuclear cells from these individuals did not transduce CCR5 signals and were highly resistant to infection in vitro with M-tropic HIV strains. Phenotypically these individuals were reported to be normal and without any immune defect. In 2,741 HIV-infected individuals from cohorts of homosexual men, hemophiliacs, and intravenous drug users from three different studies, there were no individuals with a homozygous 32 deletion (7). Recently however, an individual of European descent from among 265 HIV-infected Australian patients was found to be homozygous for 32 (2).

Because homozygous 32 confers strong protection from infection, it was surmised that the presence of this deletion may provide some survival advantage, and many studies have examined the relationship of the presence of a single mutant allele with the rate of disease progression (11). In our study cohort, the frequency of wt/32 did not differ significantly between groups of children with different rates of disease progression, although there was a trend towards higher frequency in children who survived longer than 8 years after acquisition of infection and therefore were non-rapid progressors compared to those who died or were in disease category C3 by 2 years of age. Thus, in our study the presence of a heterozygous 32 allele appeared to provide a modest degree of protection from rapid disease progression. These results are similar to those of published studies in which 32 appears to provide some protection from development of severe HIV disease in adults (11). Interestingly, we did not find CCR5 in any of the long-term survivors in our population, indicating that the modification in the disease course achieved by a single allele is modest at best. In studies of homosexual cohorts, the frequency of wt/32 in the long-term nonprogressors was found to be twice that in rapid progressors (7). In hemophiliac cohorts, on the other hand, the difference between the frequencies of the 32 allele in the two groups was not significant (7). This differential response may be related to differences in routes of transmission, exposure levels, or viral loads in different risk groups.

In summary, a relatively low frequency (4.8%) of the 32 mutation was observed in a small cohort of HIV-infected children and adolescents and was similar to the known frequency in the general nonwhite U.S. population. None of the infected children had the homozygous deletion. Children with wt/32 had a relatively favorable disease course, but none of the children in our cohort who were truly long-term nonprogressors had this deletion, indicating that other genetic host or viral factors may be important in disease pathogenesis in this population. Study of polymorphism of CCR5 and other genes in large cohorts of long-term survivors and in HIV-exposed uninfected children are warranted to determine the roles of such genetic mutations in protection from vertical infection and/or disease progression in infants with perinatal HIV exposure.