Polymorphisms of several genes, including those localized in major histocompatibility complex, have been proposed as candidates to explain the susceptibility to HIV vertical transmission in children and infants [1–5].
Several studies indicated an involvement of the nonclassical human leukocyte antigen-G (HLA-G) polymorphisms in the risk of HIV infection [6,7] and mother-to-child transmission [8–11]. Aikhionbare et al.  described an association between mother–child discordant HLA-G nucleotide sequences located at 3′untraslated region (3′ UTR), including 3777G>C (rs1710) and 14-bp deletion/insertion (D/I; rs16375) polymorphisms, and a decreased risk of HIV vertical transmission. Polymorphisms located at the 3′ UTR of the HLA-G gene may affect mRNA stability and HLA-G expression [9,12–15]; recent studies showed that the presence in homozygosis of the 14-bp insertion is associated with a very low mRNA expression of a membrane-bound isoform (HLA-G3) in placenta  and that alleles carrying the 14-bp insert sequence are associated with a lower mRNA expression in placenta samples . Moreover, Yie et al.  identified a polymorphism at the 3′ UTR of the HLA-G gene (c.1754G>A) that affects HLA-G mRNA stability by reducing the mRNA half-lives. HLA-G altered expression may influence its biological defensive action, and consequently impair its protective effect towards the fetus.
An association between HLA-G 14-bp deletion/insertion polymorphism and HIV vertical transmission in Brazilian children has been recently reported , and the deletion allele in homozygosis was found to be protective against HIV perinatal transmission.
We investigated the association between 3′ UTR HLA-G polymorphisms and perinatal transmission in two groups of Brazilian children born to HIV-positive mothers enrolled at the IMIP (Insituto de Medicina Integrada, Professor Fernando Figueira) of Recife (Brazil). One group included 163 HIV-1 perinatally infected children (80 men/83 women, mean age 6.7 ± 4.7 years), and a second group including 64 exposed uninfected children (31 men/33 women, mean age 5.6 ± 3.6 years) born to HIV-1-positive mothers who did not undergo any antiretroviral therapy or caesarean section in order to prevent vertical HIV-1 transmission. A third control group included 170 uninfected children (81 men/89 women, mean age 7.3 ± 4.6 years), from the same ethnic background of the patients and matched for sex and age. The study was approved by the IMIP Ethical Committee (CONEP 25000.127120/2001-73).
3777G>C polymorphism was analyzed by PCR  and Pst I (from Providencia stuartii) enzyme digestion, and the results obtained were double-checked by direct sequencing; allele and genotype frequencies were calculated by direct gene counting. Because the 14-bp deletion/insertion polymorphism that we previously studied  is located 35-bp upstream the 3777G>C, we performed a haplotype analysis, using Arlequin software (version 3.1) , aiming to investigate the presence of linkage disequilibrium and a possible combined effect of the two polymorphism in HIV vertical transmission. Differences in allelic, genotype and haplotype frequencies were evaluated by χ2 or Fisher's exact test. Bonferroni's correction was performed (P<0.0023 were considered statistically significant).
Allele and genotype frequencies observed for HLA-G 3777G>C polymorphism are summarized in Table 1. The genotype frequencies respected the Hardy–Weinberg equilibrium in all groups studied. We did not find any difference in the distribution of allele and genotype frequencies among the three groups analyzed. The 3777G alleles were approximately 40% and 3777C alleles were 60% in all three groups of children. The C/C genotype was slightly more frequent in HIV-positive children (41%) than in exposed uninfected (38%) and healthy controls (33%), whereas the G/C genotype was found to be more frequent in healthy controls with respect to HIV-positive children (52% versus 41%), even though this difference did not reach statistical significance after Bonferroni correction (P = 0.03).
Then we computed haplotypes and combined genotypes for the 3777G>C and 14-bp deletion/insertion polymorphism previously analyzed , aiming to investigate the combined effect of both polymorphisms on HIV vertical transmission. The results are reported in Table 1.
Haplotype analysis showed that the 14-bp deletion/insertion and the 3777G>C polymorphisms are in linkage disequilibrium (P < 0.0001 for healthy controls and HIV-positive children, P = 0.0021 for HIV-exposed uninfected, D' values = 0.94, 0.82 and 0.78, respectively). The two polymorphisms combine to form four different haplotypes, in which the D allele can associate with both C and G alleles (on average 40% C versus 60% G), whereas I allele is preferentially associated with the C (on average 95% C versus 5% G).
We found that haplotype frequencies were significantly different between HIV-exposed uninfected children and HIV-positive or healthy controls (P < 0.001 in both cases). The frequency of the 14-bp deletion/insertion D –3777C (DC) haplotype was higher in exposed uninfected children (40%) when compared to HIV positive (23%) and healthy controls (18%); conversely, the 14-bp deletion/insertion I –3777C (IC) haplotype was significantly more frequent in HIV-positive children than in HIV exposed uninfected (39% versus 20%, P < 0.001). The DC haplotype associates with a protective effect from HIV vertical transmission [odds ratio (OR) = 0.44; 95% confidence interval (CI) = 0.28–0.71].
Notably, the 3777C allele, which has similar overall frequencies in the three groups of children analyzed, is more frequently associated with the D allele (65% versus 35% I) in HIV-exposed uninfected children, whereas, in HIV-positive children and healthy controls, it is more frequently associated with the I allele (68% I versus 32% D in HIV-positive children and 64% I versus 32% D in healthy controls).
When analyzing combined genotypes, the DC/DC genotype was significantly more frequent in HIV exposed uninfected children than in healthy controls (P < 0.001) and HIV-positive children, although, in the latter case, the statistical significance is not retained after Bonferroni correction. The presence of the DC/DC combined genotype is associated with protection against HIV vertical transmission (OR = 0.22, 95% CI = 0.08–0.58).
In conclusion, we confirm the existence of a linkage disequilibrium between the 14-bp deletion/insertion and the 3777G>C polymorphisms, at 3′ UTR of HLA-G, as expected for nucleotide variations 35-bp distant from each other. In addition, we demonstrated that the 3777G>C polymorphism alone has no effect on HIV vertical transmission but, when linked with the D allele, exerts a positive role in the protection. Indeed, we identified the DC HLA-G haplotype as being significantly associated with a protective effect against HIV vertical transmission.
This study was supported by a grant from IRCCS Burlo Garofolo RC03/04; A.F. and L.S. are recipients of post doc fellowships from IRCCS Burlo Garofolo. L.S. is recipient of a fellowship grant (APQ-0020-4.01/08) from ‘Fundação de Amparo à Ciência e Tecnologia do Estado de Pernambuco’ (FACEPE). The authors wish to thank Alfredo Garzino-Demo for critically revising the article.
Authors' contribution: Ludovica Segat contributed to study design and performed data analysis and statistics; Eulalia Catamo performed the DNA extraction and HLA genotyping; Annalisa Fabris contributed to data analysis and statistics; Lara Padovan provided technical support for HLA genotyping; Marcello Morgutti performed the genotype-phenotype correlations; Sergio Crovella conceive the study design, redacted the article and contributed to genotype–phenotype correlation.
1. Farquhar C, Rowland-Jones S, Mbori-Ngacha D, Redman M, Lohman B, Slyker J, et al
. Human leukocyte antigen (HLA) B*18 and protection against mother-to-child HIV type 1 transmission. AIDS Res Hum Retroviruses 2004; 20:692–697.
2. Winchester R, Chen Y, Rose S, Selby J, Borkowsky W. Major histocompatibility complex class II DR alleles DRB1*1501 and those encoding HLA-DR13 are preferentially associated with a diminution in maternally transmitted human immunodeficiency virus 1 infection in different ethnic groups: determination by an automated sequence-based typing method. Proc Natl Acad Sci USA 1995; 92:12374–12378.
3. Winchester R, Pitt J, Charurat M, Magder LS, Goring HH, Landay A, et al
. Mother-to-child transmission of HIV-1: strong association with certain maternal HLA-B alleles independent on viral load implicates innates immune mechanisms. J Acquir Immune Defic Syndr 2004; 36:659–670.
4. Kuhn L, Abrams EJ, Palumbo P, Bulterys M, Aga R, Louie L, et al
. Perinatal AIDS Collaborative Transmission study: maternal versus paternal inheritance of HLA class I alleles among HIV-infected children – consequences for clinical disease progression. AIDS 2004; 18:1281–1289.
5. Polycarpou A, Ntais C, Korber BT, Elrich HA, Winchester R, Krogstad P, et al
. Ariel Project: association between maternal and infant class I and II HLA alleles and of their concordance with the risk of perinatal HIV type 1 transmission. AIDS Res Hum Retroviruses 2002; 18:741–746.
6. Matte C, Lajoie J, Lacaille J, Zijenah LS, Ward BJ, Roger M. Functionally active HLA-G polymorphism are associated with the risk of heterosexual HIV-1 infection in Africa women. AIDS 2004; 18:427–431.
7. Lajoie J, Hargrove J, Zijenah LS, Humphrey JH, Ward BJ, Roger M. Genetic variants in nonclassical major histocompatibility complex class I human leukocyte antigen (HLA)-E and HLA-G molecules are associated with susceptibility to heterosexual acquisition of HIV-1. J Infect Dis 2006; 193:298–301.
8. Aikhionbare FO, Hodge T, Kuhn L, Bulterys M, Abrams EJ, Bond VC. Mother-to-child discordance in HLA-G exon 2 is associated with a reduced risk of perinatal HIV-1 transmission. AIDS 2001; 15:2196–2198.
9. Aikhionbare FO, Kumaresan K, Shamsa F, Bond VC. HLA-G DNA sequence variants and risk of perinatal HIV-1 transmission. AIDS Res Ther 2006; 3:28.
10. Matte C, Zijenah LS, Lacaille J, Ward B, Roger M. Mother-to-child human leukocyte antigen G concordance: no impact on the risk of vertical transmission of HIV-1. AIDS 2002; 16:2491–2494.
11. Fabris A, Catamo E, Segat L, Morgutti M, Arraes LC, de Lima Filho JL, Crovella S. Association between HLA-G 3′UTR 14-bp polymorphism and HIV vertical transmission in Brazilian children. AIDS 2009; 14:177–182.
12. Hviid TV. HLA-G in human reproduction: aspects of genetics, function and pregnancy complications. Hum Reprod Update 2006; 12:209–232.
13. Yie S, Li L, Xiao R, Librach CL. A single base-pair mutation in the 3′-untraslated region of HLA-G mRNA is associated with preeclampsia. Mol Hum Reprod 2008; 14:649–653.
14. O'Brien M, McCarthy T, Jenkins D, Paul P, Dausset J, Carosella ED, et al
. Altered HLA-G transcription in preeclampsia is associated with allele specific inheritance: possible role of the HLA-G gene in susceptibility to the disease. Cell Mol Life Sci 2001; 58:1943–1949.
15. Hviid TV, Hylenius S, Rorbye C, Nielsen LG. HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels. Immunogenetics 2003; 55:63–79.
16. Lin A, Yan WH, Xu HH, Tang LJ, Chen XF, Zhu M, Zhou MY. 14-bp deletion polymorphism in the HLA-G gene is a risk factor for idiopathic dilated cardiomyopathy in a Chinese Han population. Tissue Antigens 2007; 70:427–431.
17. Excoffier L, Laval G, Schneider S. Arlequin ver 3.0: an integrated software package for population genetics data analysis. Evol Bioinform Online 2005; 1:47–50.