Increased frequency of the tumor necrosis factor--308 a allele in adults with human immunodeficiency virus dementia.код для вставкиСкачать
Increased Frequency of the Tumor Necrosis Factor-␣-308 A Allele in Adults With Human Immunodeficiency Virus Dementia Michael W. Quasney, MD, PhD,1,2 Qing Zhang, BS,2 Susie Sargent, MD,3 Margaret Mynatt, BS,4 Jonathan Glass, MD,5 and Justin McArthur, MD6 Genetic polymorphisms in the regulatory regions of various cytokine genes influence the amount of cytokine produced in response to inflammatory stimuli. To determine whether such polymorphisms might play a role in human immunodeficiency virus (HIV) dementia, a disease process in which tumor necrosis factor (TNF)-␣ is believed to play a role, we analyzed HIV-infected adults with and without dementia and control populations for a polymorphic site located in the promoter region of the gene coding for TNF-␣. The presence of the A allele at the TNF-␣-308 site was overrepresented among adults with HIV dementia compared to those without dementia (0.28 vs 0.07; OR 5.5; 95% CI 1.8 –17.0) and a healthy control population (0.28 vs 0.11). The increased frequency of the A allele in HIV-infected adults with dementia suggests that this locus may play a role in the pathophysiology of dementia and suggests a genetic predisposition for the development of HIV dementia. Ann Neurol 2001;50:157–162 Acquired immunodeficiency syndrome (AIDS) dementia is one of the most devastating complications of infection with the human immunodeficiency virus (HIV). It is considered the most frequent form of dementia in young adults in the United States.1 Patients exhibit a variety of cognitive and motor dysfunctions, including psychomotor slowing, memory impairment, behavioral dysfunction, and ataxia.2– 6 Although the etiology of the dementia is unclear, specific pathological and histological changes are present in the central nervous system (CNS). These include diffuse damage to white matter, multinucleated giant cells, astrocyte proliferation, microglial nodules, and macrophage infiltration.7–9 Damaged dendrites, neuronal loss in the neocortex and subcortical brain, and a loss of synapses have also been documented in brain specimens of AIDS patients.10 –16 The pathophysiology of HIV dementia remains unknown, although interactions between viral products and mediators from macrophage and microglia are believed to be involved directly in the neuronal damage.17–22 Indeed, the severity of the dementia appears to correlate best with the degree of macrophage infiltration and glial cell activation and not with viral load.23,24 Potential mediators that are neurotoxic include various cytokines, and some investigators have proposed that cytokines are the major cause of the neuropathology observed in HIV infection.20,25,26 Supporting this role for cytokines is the observation that cytokine levels are elevated in brain and cerebral spinal fluid (CSF) in HIV-infected patients with dementia compared to HIV-infected patients without dementia.21,27,28 Tumor necrosis factor-␣ (TNF-␣) in particular may play a direct role in the neuropathology in HIV dementia because of its direct toxic effects on neurons and oligodendrocytes.29 –31 Indeed, TNF-␣27,32 and TNF-␣ mRNA25 are elevated in the brains of HIV-infected patients with dementia. The stimulus for the increased amounts of TNF-␣ mRNA observed in the brains of HIV-infected adults with dementia remains unknown, although the viral proteins Tat, gp160, and gp120 are suspected.25,33–38 Several genetic polymorphisms have been found within the promoter region of the TNF-␣ gene that are associated with elevated TNF-␣ production,39 – 41 including a G to A transition at the ⫺308 site (Fig 1). The presence of an adenine at the ⫺308 site is associated with elevated TNF-␣ production in response to endo- From the 1Division of Critical Care, Department of Pediatrics, University of Tennessee, Memphis; 2Crippled Children’s Foundation Research Center, Le Bonheur Children’s Medical Center, Memphis; 3 Department of Medicine, University of Tennessee, Memphis; 4Le Bonheur Children’s Medical Center, Memphis, TN; 5Department of Neurology, Emory University School of Medicine, Atlanta, GA; and 6Department of Neurology, Johns Hopkins Hospital, Baltimore, MD. Received Nov 15, 2000, and in revised form Mar 6, 2001. Accepted for publication Mar 6, 2001. Published online May 16, 2001. Address correspondence to Dr Quasney, Division of Critical Care, Department of Pediatrics, Crippled Children’s Foundation Research Center, Le Bonheur Children’s Medical Center, 50 N. Dunlap, Memphis, TN 38103. E-mail: email@example.com © 2001 Wiley-Liss, Inc. 157 Fig 1. Location of the polymorphic sites affecting the TNF-␣ response. The more frequent allele at the TNF␣-308 site is a guanine, and the less frequent allele is an adenine. Light grey boxes ⫽ exons; dark gray boxes ⫽ introns; hatched area ⫽ promotor; white boxes ⫽ 5⬘- and 3⬘-untranslated regions. toxin compared to the more common G allele.39 Thus, there appears to be a genetic propensity to secrete higher amounts of TNF-␣. We tested the hypothesis that adults with HIV dementia may have a higher frequency of the A allele associated with elevated levels of TNF␣ at the TNF-␣-308 site. Patients and Methods Patients Brain autopsy specimens from HIV-infected adults with and without dementia were obtained from at the AIDS Brain Bank at Johns Hopkins University (Baltimore, MD). Patients were evaluated for dementia within 90 days prior to their death. Neurological and neuropathological abnormalities were defined using the American Academy of Neurology criteria42 and included (1) HIV-1 seropositivity, (2) progressive cognitive and behavioral decline, (3) neurological and/or neuropsychological findings consistent with a decline from premorbid baseline levels, and (4) CNS opportunistic process excluded by computed tomography or magnetic resonance imaging and CSF analysis. The severity of neurologic disease was scored using the Memorial Sloan-Kettering (MSK) criteria.43 HIV-seropositive, dementia-negative control samples were obtained from two sources: brain specimens from the AIDS Brain Bank and adults infected with HIV without dementia who were recruited through the Adult Special Care Clinic at the University of Tennessee, Memphis. Informed consent was obtained from patients, and human experimentation guidelines of the U.S. Department of Health and Human Services and the University of Tennessee were followed. The control populations consisted of healthy adult volunteers with no history of HIV infection, neurologic or autoimmune diseases, or chronic treatment with antiinflammatory agents. using the Genomic DNA Purification Kit (Promega, Madison, WI). The region containing the TNF-␣-308 locus was amplified using the primers TNFA-308-1 (5⬘AGGCAATAGGTTTTGAGGGCCAT-3⬘) and TNFA-308-2 (5⬘-ACACTCCCCATCCTCCCTGCT-3⬘).40 The TNFA308-1 primer contains 4 bp of the NcoI recognition sequence, including a mismatched cytosine as shown by the C in the TNFA-308-1 primer sequence. This mismatched cytosine allows for creation of an NcoI restriction site (CCATGG) when the G allele is present at position ⫺308. A 116 bp polymerase chain reaction (PCR) product was generated using the following reaction conditions: 35 cycles of denaturation at 95°C for 30 seconds, annealing at 64°C for 15 seconds, and extension at 74°C for 15 seconds. The amplified DNA was digested with NcoI, and the fragments were analyzed by electrophoresis in a 3% agarose gel and visualized by ethidium bromide staining. Interpretation was as follows: A single band at 116 bp identified individuals homozygous for an adenine at the TNF-␣-308 locus; two bands at 96 and 20 bp identified individuals homozygous for a guanine at the TNF-␣-308 locus; three bands at 116, 96, and 20 bp identified individuals heterozygous at the TNF-␣-308 locus. Statistical Analysis Statistical analysis of genotypic and allelic frequencies was performed using Fisher’s exact test. Univariate logistic analysis was performed to obtain the odds ratio between the genotype and the dependent variable of HIV infection with dementia. Statistical analysis was performed using the statistical software package GB-Stat (New England Software, Inc., Greenwich, CT). Results Patient Characteristics Sixteen HIV-infected adults with dementia, 45 HIVinfected adults without dementia, and 231 healthy adult controls were evaluated (Table 1). There were no racial differences among the 3 groups of samples analyzed. Genotypic and Allelic Frequencies at the TNF-␣-308 Site The genotypic frequencies of the TNF-␣-308 polymorphic site located in the promoter region of the gene coding for TNF-␣ were compared between HIVTable 1. Characteristics of HIV-Infected Adults With and Without Dementia and Healthy Controls Genotypic Analysis For analysis of brain samples, brain tissue was thawed, and approximately 25 mg of tissue was minced. DNA was extracted using the QIAamp Tissue Kit (Qiagen, Inc., Chatsworth, CA). For analysis of blood samples obtained from HIV-infected nondemented patients and healthy controls, whole blood (1.0 ml) was collected, and DNA was extracted 158 Annals of Neurology Vol 50 No 2 August 2001 Total Race (% Caucasian) Controls HIV ⫹ Dementia⫺ HIV ⫹ Dementia⫹ 231 0.45 45 0.47 16 0.56 HIV-infected adults with dementia compared to healthy controls, p ⫽ 0.4453; HIV-infected adults with dementia compared to HIV infected adults without dementia, p ⫽ 0.57. Statistical analysis using Fisher’s exact test. infected adults with and without dementia and a healthy control population (Table 2). There were significantly more heterozygotes in the group of HIVinfected adults with dementia than in either the group of patients with HIV-infection without dementia (0.56 vs 0.13; p ⫽ 0.002; OR 10.3; 95% CI 2.6 –39.9) or the healthy control group (0.56 vs 0.19; p ⫽ 0.004; OR 4.9; 95% CI 1.7–13.8). There were no individuals with the A/A genotype at the TNF-␣-308 site in either of the HIV-infected groups. The frequencies of the A and G alleles at the TNF-␣-308 site in HIV-infected adults with dementia were also significantly higher compared to either the nondemented group or the healthy controls (Table 2; A allele: 0.28 vs 0.07; p ⬍ 0.003; G allele: 0.28 vs 0.11; p ⬍ 0.012). There were no differences in the genotypic or allelic frequencies between those samples obtained from brain vs blood in the group of patients with HIV-infection without dementia. The allelic frequencies for the healthy control population were similar to those in previous reports.39,41,44 Discussion Several factors affect the degree of the inflammatory reaction produced by a host. These include the type of inflammatory stimulus, dose of the stimulus, and genetic characteristics of the host. In this study, HIVinfected adults with dementia were more likely to have the A allele at the TNF-␣-308 site compared to either HIV-infected adults without dementia or a healthy control population. This allele is associated with higher TNF-␣ secretion in response to an inflammatory stimulus than that in individuals with the G allele. Thus, development of dementia in HIV-infected adults may be related to the amount of TNF-␣ secreted in the brain, which in turn may be influenced by individual’s genetic predisposition to secrete higher amounts of TNF-␣. The stimulus for the increased CNS levels of TNF-␣ mRNA and TNF-␣ observed in the brains of HIVinfected adults with dementia remains unknown, although the viral proteins Tat, gp160, and gp120 are suspected.25,33–38 Exposure of human astrocytes to Tat has been shown to result in prolonged production of cytokines, including TNF-␣. However, little is known about genetic variability in the TNF-␣ response to these stimuli. Current work in our laboratory is investigating whether genetic factors influence the degree of the TNF-␣ response in the setting of infection with HIV. Evidence supports a role for TNF-␣ in neuronal damage. TNF-␣ has been shown to damage human brain cells and disrupt normal neuronal cell physiology in a number of studies. For example, TNF-␣ is neurotoxic to human cortical neurons,21 potentiates glutamate receptor-mediated neurotoxicity,45 disrupts Na⫹/K⫹ transport in astrocytes,46 disrupts glutamate uptake by astrocytes,47 and damages oligodendrocytes and myelin in vitro.29 The presence of the A allele at the ⫺308 site has been shown to result in elevated TNF-␣ production with concanavallin A41 and endotoxin.39 Polymorphic sites at positions ⫺1,031, ⫺863, ⫺857, and ⫺238 relative to the TNF-␣ transcriptional start site also have been demonstrated to have elevated TNF-␣ levels after in vitro stimulation.41 We chose to study the ⫺308 site because of the strong association of the A allele with poor outcome following other infections. For example, an association between serum TNF-␣ levels and mortality in meningococcal meningitis has been observed,48 and children with the A allele at the TNF-␣308 site have a worse outcome following meningococcal infection.49 Similar associations have been observed in individuals with malaria; those with higher TNF-␣ levels had a worse outcome,50 and the TNF-␣-308 A allele site was overrepresented among patients with cerebral malaria and among those who died from malaria.51 These previous studies prompted the present study because of the previous demonstration of elevated mRNA levels of TNF-␣ in the brains of HIVinfected adults with dementia.25 We believe that the results of this study warrant further analysis of HIVinfected patients to delineate better the association between the “high-secretor” A allele and dementia. Table 2. Genotypic and Allelic Frequencies at the TNF-␣-308 Site in HIV-Infected Adults With and Without Dementia Compared to Healthy Control Populations Genotypic Frequency Allelic Frequency Population n A/A A/G G/G A G Healthy controls HIV-infected without dementia HIV-infected with dementia 231 45 16 0.02 0.00 0.00 0.19 0.13 0.56a 0.79 0.87 0.44 0.11 0.07 0.28b 0.89 0.93 0.72 HIV-infected adults with dementia compared to HIV-infected adults without dementia, p ⫽ 0.0023; OR 7.7; 95% CI 2.1–28.7; HIV-infected adults with dementia compared to healthy controls, p ⫽ 0.0035; OR 4.8; 95% CI 1.7–13.5. b HIV-infected adults with dementia compared to HIV-infected adults without dementia, p ⫽ 0.0034; OR 5.5; 95% CI 1.8 –17.0; HIVinfected adults with dementia compared to healthy controls, p ⫽ 0.0115; OR 3.0; 95% CI 1.3– 6.9. Statistical analysis using Fisher’s exact test. a Quasney et al: TNF-␣ Polymorphisms in HIV Dementia 159 A previous report did not find an association between the high secretor allele at the TNF-␣-308 site and HIV encephalitis and/or HIV leukoencephalopathy.52 However, our study differs in that we compared brain samples HIV-infected patients with clinically demonstrated dementia to DNA extracted from HIVinfected patients without dementia. In contrast, the previous study evaluated brain samples that were histologically classified as demonstrating either HIV encephalitis or HIV leukoencephalopathy or both. No mention is made of whether these patients exhibited clinical signs of dementia. Thus, although the previous report concludes that there is no association of the high secretor allele with HIV encephalitis and/or HIV leukoencephalopathy, we conclude that an association exists between the presence of a “high secretor” allele and HIV dementia. We found an association between the frequency of the A allele at the TNF-␣-308 locus and the presence of dementia in HIV-infected adults. This suggests the possibility that the TNF-␣ induced neuronal injury observed in HIV dementia is the result of a local inflammatory reaction in individuals genetically predisposed to have a more vigorous inflammatory response. It also raises the possibility of using polymorphic analysis to identify HIV-infected individuals at risk for the development of dementia and for therapies to reduce TNF-␣ secretion to prevent neuronal injury. Before such an approach could be recommended, much larger numbers of patients with and without dementia must be studied to assess more accurately the association between the TNF-␣-308 allele and the development of dementia in HIV infection. The mechanisms by which the polymorphic sites at position TNF-␣-308 affect TNF-␣ levels are unknown. The polymorphism at the TNF-␣-308 site lies within the promoter region for the TNF-␣ gene and may alter the binding of transcription factors. Studies with the TNF-␣ promoter linked to a chloramphenicol acetyltransferase reporter gene have demonstrated that the A allele at TNF-␣-308 site is associated with higher constitutive and inducible levels of transcription than the more common G allele.53 Kroeger, et al,54 demonstrated, furthermore, a variability in the ability of this region to bind nuclear proteins depending on a single nucleotide difference in the oligonucleotide. Current studies are continuing in our laboratory to identify the molecular mechanisms by which this region affects serum levels of TNF-␣. There are some limitations to our study. First, it may be that the precise genetic variation associated with dementia in HIV-infected adults is not at the TNF-␣-308 site but rather at one closely linked to it. The gene for TNF-␣ is located in the MHC complex, raising the possibility that the MHC type or another closely linked gene is more directly responsible for the 160 Annals of Neurology Vol 50 No 2 August 2001 dementia. Second, a possibility exists that tissuespecific genetic variations at the TNF-␣-308 site are present in the brain and blood samples and that our observed differences are the result of these tissuespecific differences. However, several of the samples in the group of HIV-infected adults without dementia were obtained from brain specimens, and there were no differences in the genotypic or allelic frequencies between those samples obtained from brain vs blood in this group. Finally, the cohort of HIV-infected patients with dementia is small. We suggest that a larger prospective study evaluating the association of TNF-␣ polymorphisms with dementia in HIV-infected adults is warranted. In summary, HIV-infected adults with dementia have a higher frequency than HIV-infected adults without dementia or a healthy control group of the A allele at the TNF-␣-308 site in the promoter of the gene coding for TNF-␣. These findings suggest that the development of dementia in adults with HIV may be in part influenced by genetic factors. These findings also suggest the possibility of identifying HIV-infected adults who may be at risk for the development of dementia and for whom anti-TNF-␣ strategies may be developed to lessen this risk. Further studies are needed to test the hypothesis supported by this study that genetic polymorphisms that influence the host immune response may play an important role in HIV dementia. This project was supported in part by the Crippled Children’s Foundation (M.W.Q.) and NIH grant NS26643 ( J.M.). References 1. Bacellar H, Munoz A, Miller EN, et al. 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