Abstract
Apoptosis has been suggested as a major mechanism for the CD4+ T-lymphocyte depletion observed in patients infected with human immunodeficiency virus 1 (HIV-1). To evaluate the impact of genetic variations to apoptosis during progression of acquired immunodeficiency syndrome (AIDS), we have performed an extensive genetic analysis of Fas and Fas ligand (FasL) genes. The coding regions and promoters of these genes were resequenced in a cohort of 212 HIV-1-seropositive patients presenting extreme disease phenotypes and 155 healthy controls of Caucasian origin. Overall, 33 single nucleotide polymorphisms (SNPs) with an allele frequency >1% were identified and evaluated for their association with disease progression. Among them, 14 polymorphisms were newly characterized. We did not find any statistically significant association of Fas and FasL polymorphisms and haplotypes with AIDS progression.
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Apoptosis, or programmed cell death, is a key mechanism allowing multicellular organisms to tightly regulate cell growth, preventing pathological processes such as autoreactivity, cancer and immunodeficiency. In the pathogenesis of acquired immunodeficiency syndrome (AIDS), apoptosis is considered to play an important role in human immunodeficiency virus 1 (HIV-1)-dependent CD4+ T-lymphocyte depletion, induced and accelerated by the virus itself (Ameisen and Capron 1991; Gougeon et al. 1991; Laurent-Crawford et al. 1991; Terai et al. 1991; Westendorp et al. 1995; Zinkernagel 1995). Indeed, earlier studies showed a great fragility of CD4+ T cells leading to their apoptosis in patients infected with HIV-1 (Ameisen and Capron 1991; Groux et al. 1992). It was shown later that the degree of CD4+ T-cell apoptosis in patients prior to T-cell depletion tightly correlates with disease progression (Gougeon et al. 1996; Liegler et al. 1998).
Several different extrinsic pathways are hypothesized to induce death receptor-associated apoptosis in AIDS. Among them, accumulating evidences strongly indicate the direct involvement of the Fas/Fas ligand (FasL) pathway to the T-lymphocyte depletion. In T lymphocytes of HIV-1-positive individuals, the expression of Fas and FasL is significantly increased (Debatin et al. 1994; Mitra et al. 1996) and Fas-mediated apoptosis is accelerated by antibody cross-linking (Katsikis et al. 1995). Upregulated expression of FasL in macrophages infected with HIV-1 can induce apoptotic death of normal peripheral blood T cells in vitro, suggesting another death-signal transmission through macrophages in T-cell depletion (Badley et al. 1996). Moreover, a study using anti-Fas antibodies showed a significant decrease of Fas-induced cell death in T cells of patients with extremely slow progression as well as in one of their uninfected parents (Bottarel et al. 2001). This indicates the inherited alteration of the Fas-signaling pathway resulting in the quantitative difference in the degree of apoptosis, and thus the disease progression.
In the present study, we focused on genetic variations of Fas and FasL genes to clarify whether genetic variations of these apoptosis-related genes could influence disease onset and progression. To perform this study, we employed the GRIV cohort consisting of two sub-populations of Caucasian HIV-1-positive individuals with extreme phenotypes: 154 asymptomatic individuals (SP) with a CD4+ cell count above 500/mm3 for 8 or more years after seroconversion and 58 patients with rapid progression (RP) showing a drop in their CD4+ cell count below 300/mm3 in less than 3 years after the last seronegative test (for more details, see Flores-Villanueva et al. 2003; Huber et al. 2003; Rappaport et al. 1997). We also employed 155 seronegative control (CTR) subjects from the same ethnic origin.
We systematically screened the Fas and FasL genes for polymorphisms by resequencing the exons, flanking regions and promoter regions. We identified 46 and 30 polymorphisms in Fas and FasL genes, respectively (Fig. 1). Out of these 76 polymorphisms identified, 33 single nucleotide polymorphisms (SNPs) have an allele frequency of 1% or greater in at least one of SP, RP and CTR groups. Fourteen among these 33 SNPs are newly identified in this study. As shown in Fig. 1, 6 SNPs are located in exons. The SNPs Fas_12063 and Fas_12037 introduce synonymous changes, while the other four are located either in the 5′UTR (Fas_12013) or the 3′UTR (Fas_12057, Fas_12066 and Fas_12075). Table 1 summarizes the frequency of each SNP in the SP, RP and CTR populations, their association with AIDS progression and the relevant information known to date for each SNP. The SNP distributions in cases and controls were in Hardy-Weinberg equilibrium.
We performed statistical analysis to test whether these polymorphisms or deduced SNP haplotypes are associated with disease progression. We failed to obtain any significant association between polymorphisms in Fas and FasL genes and AIDS progression by Fisher’s exact test (the lowest P value is 0.096 in FasL_533 between RP and SP). Similarly, the same test using estimated haplotypes (Fig. 2) did not find any significant association between particular haplotypes of Fas or FasL genes and AIDS progression (Table 2). The lowest P-value was 0.102 for the haplotype 3 of the Fas gene obtained by comparing RP with SP.
A general case-control study using 283 HIV-positive patients and 111 control subjects (Cascino et al. 1998) on two SNPs in the Fas gene (Fas_12063 and Fas_12037) did not show significant association between combination of two SNPs and AIDS. The more specific study shown here using two sub-populations of AIDS patients with extreme phenotypes did not provide any further genetic evidence that Fas and FasL genes are major genetic determinants in the onset and pathogenicity of HIV-1 infection.
Increased apoptosis of T cells has been reported after Epstein-Barr virus infections (Akbar et al. 1993; Uehara et al. 1992), varicella-zoster infections (Akbar et al. 1993) and infections induced by herpesviridae (Razvi and Welsh 1993). So the identification of 14 novel SNPs with relatively high frequency (>1%) and haplotype information on these genes should prove useful for the genetic study of patients with other virus infections or with cancer. In another hand, more extensive genetic studies on additional genes related to Fas/FasL-mediated apoptosis will clarify the role of this pathway in AIDS.
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Acknowledgements
The authors are grateful to all the patients and all the medical staff who have generously collaborated with the GRIV project. The authors also thank the EGEA cooperative group for having given access to data on the EGEA (Epidemiological Study on the Genetics and Environment of Asthma), which was partly supported by an INSERM/MSD convention. The GRIV project is supported by grants from ANRS (Agence Nationale de Recherche sur le SIDA), ACV (AIDS-Cancer Vaccine Development Foundation) and Neovacs SA. The CNG is supported by the Ministere de la Recherche et des Nouvelles Technologies.
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Vasilescu, A., Heath, S.C., Diop, G. et al. Genomic analysis of Fas and FasL genes and absence of correlation with disease progression in AIDS. Immunogenetics 56, 56–60 (2004). https://doi.org/10.1007/s00251-004-0664-3
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DOI: https://doi.org/10.1007/s00251-004-0664-3