HLA polymorphisms and detection of kaposi sarcoma-associated herpesvirus DNA in saliva and peripheral blood among children and their mothers in the uganda sickle cell anemia KSHV Study
© Guech-Ongey et al; licensee BioMed Central Ltd. 2010
Received: 20 September 2010
Accepted: 18 November 2010
Published: 18 November 2010
Kaposi sarcoma-associated herpesvirus (KSHV, also called Human herpesvirus 8 or HHV8) is a γ-2 herpesvirus that causes Kaposi sarcoma. KSHV seroprevalence rates vary geographically with variable rates recorded in different sub Sahara African countries, suggesting that effects of genetic and/or environmental factors may influence the risk of infection. One study conducted in South Africa, where KSHV seroprevalence is relatively low, found that carriage of human leukocyte antigen (HLA) alleles HLA-A*6801, HLA-A*30, HLA-A*4301, and HLA-DRB1*04 was associated with increased shedding of KSHV DNA in saliva. Confirmation of those results would strengthen the hypothesis that genetic factors may influence KSHV distribution by modulating KSHV shedding in saliva. To explore these associations in another setting, we used high resolution HLA-A, B, and DRB1 typing on residual samples from the Uganda Sickle Cell Anemia KSHV study, conducted in a high KSHV seroprevalence region, to investigate associations between HLA and KSHV shedding in saliva or peripheral blood among 233 children and their mothers. HLA-A and HLA-DRB1 alleles were not associated with KSHV shedding in our study, but our study was small and was not adequately powered to exclude small associations. In exploratory analyses, we found marginal association of KSHV DNA shedding in saliva but not in peripheral blood among children carrying HLA- B*4415 and marginal association of KSHV DNA shedding in peripheral blood but not in saliva among children carrying HLA- B*0801 alleles. The contribution of individual HLA polymorphisms to KSHV shedding is important but it may vary in different populations. Larger population-based studies are needed to estimate the magnitude and direction of association of HLA with KSHV shedding and viral control.
Kaposi sarcoma-associated herpesvirus (KSHV, also called Human herpesvirus 8 or HHV8) is a γ-2 herpesvirus etiologically linked to Kaposi sarcoma (KS) . KSHV seroprevalence in the general population is highest in sub-Saharan Africa (50-60%), intermediate in South America and the Mediterranean countries (10-40%), and low in Europe and North America (< 5%) , suggesting that genetic and/or environmental factors may influence KSHV seropositivity patterns [3, 4]. Environmental factors, including plants in regions of Africa where KSHV prevalence and KS incidence are high, have been postulated to influence KSHV lytic replication and, thus, increase KSHV shedding . Colluzi et al., speculated that KSHV transmission is increased indirectly when KSHV infected saliva is used to soothe itchy insect bites caused by blood-sucking arthropods . The role of genetic factors is less well understood. An effect of a major recessive gene on KSHV susceptibility or resistance to KSHV infection was postulated, based on statistical analysis of familial clustering of KSHV infection, among Noire-Marrons families in French Guiana . Recently, a study in South Africa found increased KSHV DNA shedding in saliva among subjects carrying human leukocyte antigens (HLA) HLA-A*6801, HLA-A*30, HLA-A*4301, and HLA-DRB1*04 alleles . Given that HLA polymorphisms have been shown to influence transmission, control, and pathogenesis of other viral infections such as human papillomavirus, human immunodeficiency virus type 1 and human T cell lymphotropic virus type 1 [9–11], this finding is biologically plausible. Thus, we sought to replicate the findings from South Africa in Uganda and to explore novel HLA allele associations with KSHV shedding using residual samples and data from the Uganda Sickle Cell Anemia KSHV study .
The Uganda Sickle Cell Anemia KSHV study included 600 children with sickle cell anemia, but without KS, who were enrolled from November 2001 to April 2002 at the Sickle Cell Clinic at Mulago Hospital . The mothers of the children were also included when available. Children were confirmed by gel electrophoresis to have sickle cell anemia (homozygous for the sickle cell gene) and the mothers were presumed to have the sickle cell trait (heterozygous for the sickle cell gene), but not sickle cell anemia. Children and their mothers were tested serologically for anti-KSHV antibodies using two peptide enzyme immunoassays (EIA) to the K8.1 and ORF73 KSHV peptides, as previously described . A subset of 233 children, including 183 who were KSHV seropositive on either the K8.1 or the ORF73 EIA and 50 children randomly selected from among 417 children who were seronegative on both assays, were further studied for KSHV viral shedding in saliva and in peripheral blood using quantitative polymerase chain reaction (qPCR) for KSHV DNA .
We performed high-resolution HLA-A, -B and -DRB1 typing on residual DNA obtained from saliva or buffy coat of the children and their mothers from the KSHV viral study. Alleles were separated by a group-specific amplification approach using multiple amplification primer mixes in parallel (PROTRANS S3/S4 HLA SINGLE ALLELE SEQUENCING SET, PROTRANS MEDICAL DIAGNOSTICS, HOCKENHEIM, GERMANY) . The presence or absence of a PCR product was demonstrated by an agarose gel-based read-out for HLA-A and HLA-B while the PCR product detection for HLA-DRB1 was achieved by fluorescence-based read-out using the 5' nuclease technology . Purification of the PCR product was performed enzymatically by exonuclease/shrimp alkaline phosphatase treatment (ExoSAP-IT; USB; Cleveland, OH, USA), followed by forward and reverse sequencing of exon 2 and 3 for HLA class I and of exon 2 for HLA class II, using Big Dye Terminator Technology (Applied Biosystems, Foster City, CA, USA). The sequencing reaction products were purified using the Montage SEQ96 sequencing reaction cleanup kit (Millipore, Billerica, MA, USA) and subjected to electrophoresis on a 3730 Genetic Analyzer (Applied Biosystems). The data were analyzed using the Sequence Pilot program (version 3.0; Protrans). Sequencing-based typing allowed determination of HLA allele groups on basis of resolution at the 2- digit level and alleles on basis of resolution at the 4-digit level .
We calculated the weighted distribution of HLA-A, -B, and -DRB1 allele group frequencies among the children and their mothers separately. The prevalence of allele groups was weighted back to the original study population. Thus, the weighted prevalence of HLA allele group (X) in the children = (prevalence of allele group X in KSHV-negative children × proportion of children who were KSHV negative) + (prevalence of allele group X in KSHV-positive children X proportion of children who were KSHV positive). The prevalence of HLA allele groups for the mothers was weighted back to the original study population based on KSHV status of the children because the mothers were selected when their child was selected.
We assessed associations between HLA allele groups and KSHV DNA detection for the alleles selected a priori based on previous associations with KSHV shedding in saliva: HLA-A*6801, HLA-A*30 and HLA-A*4301, and HLA-DRB1*04 separately for the children and their mothers. We also performed exploratory analyses to identify new associations. Odds ratios (ORs) for association and corresponding 95% confidence intervals (95% CIs) were computed using logistic regression models. We adjusted for KSHV serostatus by including the posterior probability of KSHV infection estimated using multivariate mixture models as previously described . All statistical tests were two-sided and p-values < 0.05 were considered statistically significant.
Weighted* HLA allele group prevalence among children and mothers in the Uganda Sickle Cell Anemia KSHV Study, 2001-2002
Percent positive for allele group (number positive)
Percent positive for allele group (number positive)
Association between HLA alleles and KSHV DNA detection in saliva and peripheral blood among children in the Uganda Sickle Cell Anemia KSHV Study, 2001-2002
# KSHV DNA positive/#KSHV DNA negative
OR (95% CI)
# KSHV DNA positive/#KSHV DNA negative
OR (95% CI)
A priori-motivated analyses
At least 1 *3001
At least *3002
At least 1*0405
At least 1*0801
At least 1*4403
1.1 (0.1- 9.9)
At least 1*4415
Our study did not replicate HLA associations with KSHV DNA shedding reported in South Africa . Our null results are likely due to the small size of our study and hence limited power to demonstrate weak associations. Another reason is that, our study included a highly selected group of children, i.e., with sickle cell anemia and their mothers with sickle cell trait, who may be systematically different in their HLA genotype distribution from the general population. In addition, because KSHV shedding is often intermittent, associations between KSHV shedding and HLA may have been missed by our study that relied on peripheral blood and saliva, taken at one time point. We found that the distributions of HLA allele groups in the mothers and children were similar and resembled the distributions of HLA allele groups in two general population-based studies in Uganda [16, 17] suggesting that the selected nature of our population may not be a major reason for our null results. Possibly, different alleles influence KSHV shedding in different populations, as suggested by the rare-allele advantage model . The polymorphic nature of HLA is critical for conferring diverse and effective pathogen response. Because polymorphisms in HLA are influenced by pathogens that are common in the environment where the population resides and is well adapted, differences in haplotype structure in populations residing in geographically dispersed regions, such as Uganda and South Africa, could lead to variable HLA pathogen associations . For example, HLA allele polymorphisms HLA- B*5301, HLA- DRB1*1302, and HLA- DRB1*0101 have been associated with genetic resistance [20–22] or HLA-DRB1*04 with susceptibility  to malaria, but the distribution is different in different populations where malaria is endemic [19, 20, 24]. In the study by, Alkharsah et al., , risk for KSHV shedding in saliva was increased among carriers of HLA-A*43, but this allele group was observed in 2.8% of women in their study. This allele group was not observed in our study and it was absent in another study that evaluated HLA polymorphisms in Uganda  and, thus, its effect cannot be replicated in Uganda. Taken together, ours and Alkharsah's study  provide the first insights about HLA associations with KSHV DNA shedding but additional studies are warranted to clarify the role of host genetics in KSHV epidemiology.
The strengths of our study include assessment of HLA polymorphisms on three loci among children and their mothers. The pre-selected sample of children with sickle cell disease, one time sampling of subjects and small sample size were limitations. Thus, it is possible that play of chance and multiple testing in small studies could explain findings from both ours and Alkharsah's studies. Large and better designed studies are warranted to evaluate the contribution of individual HLA polymorphisms to KSHV shedding in different populations.
We are grateful to the study subjects and staff at the Sickle Cell Clinic at Mulago Hospital, Kampala, for their participation. Mothers gave written consent for themselves and their children to participate; children ≥ 7 years old gave witnessed assent. The study was approved by the Institutional Review Boards of the National Cancer Institute and the Uganda National Council for Science and Technology. The content is the responsibility of the authors alone and does not necessarily reflect the views or policies of the United States Department of Health and Human Services.
This work was funded by the Intramural Research Program of the Division of Epidemiology and Cancer Genetics, National Cancer Institute (contracts N02-CP-91027 and N01-CO-12400), National Institutes of Health, Department of Health and Human Services.
- Martin JN: Diagnosis and epidemiology of human herpesvirus 8 infection. Semin Hematol. 2003, 40 (2): 133-42. 10.1016/S0037-1963(03)70005-5.PubMedView ArticleGoogle Scholar
- Dukers NH, Rezza G: Human herpesvirus 8 epidemiology: what we do and do not know. AIDS. 2003, 17 (12): 1717-30. 10.1097/00002030-200308150-00001.PubMedView ArticleGoogle Scholar
- Mbulaiteye SM: Detection of kaposi sarcoma-associated herpesvirus DNA in saliva and buffy-coat samples from children with sickle cell disease in Uganda. J Infect Dis. 2004, 190 (8): 1382-6. 10.1086/424489.PubMedView ArticleGoogle Scholar
- Dedicoat M: Mother-to-child transmission of human herpesvirus-8 in South Africa. J Infect Dis. 2004, 190 (6): 1068-75. 10.1086/423326.PubMedView ArticleGoogle Scholar
- Whitby D: Reactivation of Kaposi's sarcoma-associated herpesvirus by natural products from Kaposi's sarcoma endemic regions. Int J Cancer. 2007, 120 (2): 321-8. 10.1002/ijc.22205.PubMedPubMed CentralView ArticleGoogle Scholar
- Coluzzi M: Reduced seroprevalence of Kaposi's sarcoma-associated herpesvirus (KSHV), human herpesvirus 8 (HHV8), related to suppression of Anopheles density in Italy. Med Vet Entomol. 2003, 17 (4): 461-4. 10.1111/j.1365-2915.2003.00465.x.PubMedView ArticleGoogle Scholar
- Plancoulaine S: Evidence for a recessive major gene predisposing to human herpesvirus 8 (HHV-8) infection in a population in which HHV-8 is endemic. J Infect Dis. 2003, 187 (12): 1944-50. 10.1086/375345.PubMedView ArticleGoogle Scholar
- Alkharsah KR: Influence of HLA alleles on shedding of Kaposi sarcoma-associated herpesvirus in saliva in an African population. J Infect Dis. 2007, 195 (6): 809-16. 10.1086/511827.PubMedView ArticleGoogle Scholar
- Davidson EJ: Association between human leukocyte antigen polymorphism and human papillomavirus 16-positive vulval intraepithelial neoplasia in British women. Cancer Res. 2003, 63 (2): 400-3.PubMedGoogle Scholar
- MacDonald KS: Influence of HLA supertypes on susceptibility and resistance to human immunodeficiency virus type 1 infection. J Infect Dis. 2000, 181 (5): 1581-9. 10.1086/315472.PubMedView ArticleGoogle Scholar
- Jeffery KJ: The influence of HLA class I alleles and heterozygosity on the outcome of human T cell lymphotropic virus type I infection. J Immunol. 2000, 165 (12): 7278-84.PubMedView ArticleGoogle Scholar
- Mbulaiteye SM: Human herpesvirus 8 infection and transfusion history in children with sickle-cell disease in Uganda. J Natl Cancer Inst. 2003, 95 (17): 1330-5.PubMedView ArticleGoogle Scholar
- Verboom M: Bioinformatic integration of biomechanics makes HLA sequencing universally applicable. Tissue Antigens. 2007, 70 (4): 338-9. 10.1111/j.1399-0039.2007.00919.x.PubMedView ArticleGoogle Scholar
- Albis-Camps M, Blasczyk R: Fluorotyping of HLA-DRB by sequence-specific priming and fluorogenic probing. Tissue Antigens. 1999, 53 (3): 301-7. 10.1034/j.1399-0039.1999.530312.x.PubMedView ArticleGoogle Scholar
- Pfeiffer RM: Combining assays for estimating prevalence of human herpesvirus 8 infection using multivariate mixture models. Biostatistics. 2008, 9 (1): 137-51. 10.1093/biostatistics/kxm018.PubMedPubMed CentralView ArticleGoogle Scholar
- Kijak GH: HLA class I allele and haplotype diversity in Ugandans supports the presence of a major east African genetic cluster. Tissue Antigens. 2009, 73 (3): 262-9. 10.1111/j.1399-0039.2008.01192.x.PubMedView ArticleGoogle Scholar
- Cao K: Differentiation between African populations is evidenced by the diversity of alleles and haplotypes of HLA class I loci. Tissue Antigens. 2004, 63 (4): 293-325. 10.1111/j.0001-2815.2004.00192.x.PubMedView ArticleGoogle Scholar
- Trachtenberg E: Advantage of rare HLA supertype in HIV disease progression. Nat Med. 2003, 9 (7): 928-35. 10.1038/nm893.PubMedView ArticleGoogle Scholar
- A global network for investigating the genomic epidemiology of malaria. Nature. 2008, 456 (7223): 732-7. 10.1038/nature07632.
- Hill AV: Molecular analysis of the association of HLA-B53 and resistance to severe malaria. Nature. 1992, 360 (6403): 434-9. 10.1038/360434a0.PubMedView ArticleGoogle Scholar
- Hill AV: Common west African HLA antigens are associated with protection from severe malaria. Nature. 1991, 352 (6336): 595-600. 10.1038/352595a0.PubMedView ArticleGoogle Scholar
- Hill AV: The immunogenetics of human infectious diseases. Annu Rev Immunol. 1998, 16: 593-617. 10.1146/annurev.immunol.16.1.593.PubMedView ArticleGoogle Scholar
- Osafo-Addo AD: HLA-DRB1*04 allele is associated with severe malaria in northern Ghana. Am J Trop Med Hyg. 2008, 78 (2): 251-5.PubMedGoogle Scholar
- Ellis JM: HLA-B allele frequencies in Cote d'Ivoire defined by direct DNA sequencing: identification of HLA-B*1405, B*4410, and B*5302. Tissue Antigens. 2001, 57 (4): 339-43. 10.1034/j.1399-0039.2001.057004339.x.PubMedView ArticleGoogle Scholar
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