Abstract
Immunity against cytomegalovirus (CMV) is initiated after its recognition by Toll-like receptor 2 (TLR2). We assessed the association between a single-nucleotide polymorphism (SNP) that impairs TLR2 function and CMV disease in a cohort of 737 liver recipients. Ninety-two of 737 patients (7.1%, 10.9%, 12.3%, and 12.5% by 3, 6, 12, and 24 months, respectively) developed CMV disease. Kaplan-Meier estimation demonstrated an association between TLR2 R753Q SNP homozygosity and CMV disease (P = .044), especially tissue-invasive CMV disease (P = .001). A multivariate Cox proportional hazard model that accounted for other significant predictors demonstrated a significant association between TLR2 R753Q SNP homozygosity and tissue-invasive CMV disease (hazard ratio, 3.407; 95% confidence interval, 1.518–7.644; P = .0029). In conclusion, homozygosity for TLR2 R753Q SNP is a marker for CMV disease risk, especially for tissue-invasive disease, after liver transplantation. This observation supports the critical role of TLR2 in the pathogenesis of CMV disease in humans.
Although the incidence of and mortality associated with cytomegalovirus (CMV) disease have been markedly reduced by effective antiviral drugs for prevention and treatment, it remains an important cause of morbidity, especially during the first year after transplantation [1–5]. Severe pathogen-specific immunodeficiency during the initial period after transplantation is the major factor that determines the risk of CMV disease [6, 7]. CMV-seronegative transplant candidates who are lacking CMV-specific immunity and receive an allograft from a CMV-seropositive donor (CMV D+/R−) are at the highest risk of this infection [1, 8]. Other clinical factors that suppress or delay the generation of effective adaptive immunity, such as the use of lymphocyte-depleting immunotherapy, further enhance the risk of CMV disease after transplantation [1, 8]. There is increasing evidence to suggest that defects in innate immune molecules, such as pathogen-recognition receptors, may also predispose to a higher risk of CMV disease during this highly vulnerable period after transplantation [9–11].
The innate immune response to CMV is initiated after the recognition of its envelope glycoproteins by Toll-like receptor 2 (TLR2), a transmembrane protein that is expressed on the surface of numerous cells [12–14]. Observations from experimental models and in vitro investigations have shown that TLR2 senses the envelope glycoproteins B and H of CMV [12, 13]. This TLR2-CMV interaction initiates a cascade of intracellular signaling events that culminate in the production of antiviral peptides and cytokines and subsequently herald the generation of protective adaptive immunity [12, 13]. In vitro, CMV glycoprotein B induces cellular activation only when TLR2 is expressed on the cell surface, an effect that was not elicited in TLR2-deficient cells [9]. In vivo, mice that are deficient of TLR2 possessed very high hepatic and splenic levels of CMV [14]. We have demonstrated elsewhere the clinical relevance of these experimental data in an initial pilot study of 92 liver transplant recipients with chronic hepatitis C virus (HCV) infection [11]. Liver transplant recipients with the TLR2 R753Q single-nucleotide polymorphism (SNP), which encodes for a substitution in the amino acid glutamine for arginine at position 753 of the protein-receptor, was marginally associated with CMV disease after liver transplantation [11]. Because this initial study was conducted only in a modest and homogenous population of HCV-infected liver transplant recipients, we aimed to confirm the clinical relevance of this observation in a much larger, independent, and heterogeneous cohort of patients who received liver transplants for various indications.
MATERIALS AND METHODS
Study Population
The study cohort consisted of consecutive patients who received liver transplantation at the Mayo Clinic in Rochester, Minnesota, from January 1997 through April 2006. In compliance with Minnesota law, only patients who provided consent for the review of their medical records were included. Of the 756 eligible patients, a total of 737 were eventually included in this study; the remaining 19 patients were excluded because of lack of blood sample or research authorization. This study was approved by the Institutional Review Board of the Mayo Foundation.
Medical Record Review
The medical records of all 737 patients were reviewed for demographic and clinical characteristics, immunosuppressive regimens, acute allograft rejection, bacterial infection, and donor and recipient CMV serologic status. All patients were followed-up until the date of death or last clinic follow-up.
Immunosuppressive Regimens
Intravenous methylprednisolone was given as the induction immunosuppressive therapy at the time of liver transplantation in the vast majority of patients. Beginning in 2002, daclizumab was also used as induction therapy for certain liver transplant recipients with chronic kidney disease. In general, maintenance immunosuppressive therapy consisted of a triple regimen with prednisone, an antimetabolite (azathioprine or mycophenolate mofetil), and a calcineurin inhibitor (cyclosporine or tacrolimus). Beginning in 1999, the triple regimen consisted mainly of prednisone, mycophenolate mofetil, and tacrolimus. Our clinical practice is to discontinue mycophenolate mofetil at 2 months after liver transplantation, unless the patient meets the criteria for continuation of therapy (such as low tacrolimus level or history of treated acute allograft rejection). The dose of prednisone is gradually tapered off over the course of 4 months after liver transplantation, at which time it is discontinued, unless circumstances, such as retransplantation or acute rejection, warrant its use. In the long term, the vast majority of liver transplant recipients were maintained solely on tacrolimus monotherapy. Sirolimus was used as an alternative regimen in only a few patients.
Definition of CMV Disease
CMV disease was defined according to accepted definitions and was categorized into CMV syndrome or tissue-invasive CMV disease [15, 16]. CMV syndrome was defined as the detection of CMV in the blood, accompanied by fever, fatigue, malaise, leukopenia, thrombocytopenia, and/or arthralgias; other potential causes were excluded, and there was no evidence of tissue-invasive disease [15, 16]. Definite tissue-invasive CMV disease was defined as the presence of clinical signs and symptoms of tissue involvement, such as diarrhea, elevated liver enzyme levels, or mucosal ulcerations, accompanied by virologic and histologic detection of CMV in a biopsy specimen [15, 16]. In the absence of biopsy, patients with symptoms of organ involvement, along with a positive CMV blood culture result or CMV DNA in plasma, were classified as having probable tissue-invasive CMV disease if the specific symptoms improved after anti-CMV treatment. Both the probable and definite cases were combined in the analysis for tissue-invasive CMV disease.
Prevention of CMV Disease
CMV prevention strategies varied during the study period [17]. During 1996–2000, a randomized, double blind, placebo-controlled trial was conducted to assess the efficacy of preemptive therapy with oral ganciclovir (1 g 3 times daily for 8 weeks, adjusted on the basis of renal function) for preventing CMV disease [5, 18]. Beginning in 2000, our clinical practice has been to provide antiviral prophylaxis to all CMV D+/R− liver transplant recipients [17]. As soon as the patient is able to tolerate oral medications, antiviral prophylaxis with oral ganciclovir (1 g 3 times a day [before October 2001]) or valganciclovir (900 mg once daily [on or after October 2001]) is started and continued for 90–100 days after liver transplantation. CMV-seropositive liver transplant recipients received oral acyclovir for herpes simplex virus prevention and were monitored for CMV reactivation and treated preemptively after detection of CMV DNA in plasma. Patients who developed acute rejection received anti-CMV prophylaxis for 4 weeks after the treatment with intravenous methylprednisolone, muromonab-CD3 (OKT3), or antithymocyte globulin [19].
Detection of TLR2 R753Q SNP
Blood samples from all patients were collected before liver transplantation and were stored at −80°C in the tissue and serum bank. After sample thawing, deoxynucleic acid was extracted from the peripheral blood samples (200 μL of whole blood or 106 peripheral blood mononuclear cells) with use of the Isoquick Nucleic Acid Method (ORCA Research), according to the manufacturer’s instructions. The extracted nucleic acid was eluted in 100 μL of sterile DNase- and RNase-free water. Five microliters of the eluted DNA was used for detection of TLR R753Q SNP with use of real-time polymerase chain reaction (PCR) assay performed on a LightCycler instrument, as described elsewhere [11, 20, 21]. In brief, 5 μL of extracted DNA was mixed with 15 μL of PCR solution consisting of LightCycler FastStart master mix (Roche Molecular Biochemicals), 0.25 mmol/L of each primer (sense primer 5′-AGTGA- GCGGGATGCCTACT-3′ and antisense primer 5′-GACTTTATCGCACCTCCAGATT- TAC-3′), 4 mmol/L magnesium chloride, 9.5 mL of water, 0.2 mmol/L TLR2 sensor probe (5′- CAAGCTGCAGAAGATAATGAACACCAAG-3′-FL), and 0.4 mmol/L TLR2 anchor probe (LC Red 640–5′-CCTACCTGGAGTGGCCCATGGACG-3′). Initial denaturation was performed at 95°C for 10 min, followed by 45 cycles of denaturation (95°C for 0 s, 20°C per s), annealing (55°C for 10 s), and extension (72°C for 18 s). Melting curve analysis involved 1 cycle at 95°C for 0 s and 53°C for 30 s, followed by an increase in temperature to 80°C at a slope of 0.1°C per s. For melting curve analysis, the wild-type TLR2 had a melting peak of 61.8°C, whereas the SNP that resulted in the R753Q substitution had a melting peak of 66.3°C. The PCR results in randomly selected samples were confirmed by gene sequencing of representative samples.
Statistical Analysis
Data analysis was performed using descriptive statistics, including mean (and standard deviation), median (and ranges), and 95% confidence intervals (CIs). Kaplan-Meier estimation was used to assess the association between TLR2 R753Q SNP and CMV disease-free survival. Patients were divided into subgroups according to their CMV recipient and donor serologic status. Kaplan-Meier estimation was used to assess the association between TLR2 R753Q SNP and CMV-free survival in each subgroup. Multivariable Cox proportional hazard models in R+ and D+/R− patients were used to assess associations between covariates and CMV disease and tissue-invasive CMV disease. The covariates of treated allograft rejection and bacterial infection were considered as time-dependent covariates using categories of in an episode and outside an episode, in which the episodes lasted 90 days and 30 days, respectively. Overlapping episodes of the same type were combined. All variables significant univariately at the 0.10 level were included in the multivariable models, with the exception of TLR2 R753Q SNP, which was included in all models. In all survival analyses, patients were censored at the date of last follow-up or death. Statistical significance was set at P ≤ .05 in all final assessments.
RESULTS
Study Population
Of the 737 liver transplant recipients who were screened for the presence of the TLR2 R753Q SNP, the majority (628 [89%]) possessed the wild-type TLR2 allele. Only 81 patients (11%) had ≥1 TLR2 allele with the R753Q SNP; this included 52 patients who were homozygotes. The demographic and clinical characteristics of the entire cohort are shown in Table 1. The mean age (± standard deviation) of the patients at the time of liver transplantation was 51.9 ± 10.46 years. The 2 leading indications for liver transplantation were chronic HCV infection (23.6%) and alcoholic liver disease (21.2%). Almost all patients received induction therapy with intravenous methylprednisolone (98.4%); only 51 patients (6.9 %) received daclizumab therapy.
Table 1.
Demographic and Clinical Characteristics of 737 Liver Transplant Recipients, Mayo Clinic Rochester, 1997–2006
| Clinical and Demographic Variable | Value |
| Age, years | |
| Mean (SD) | 51.9 (10.45) |
| Median (IQR) | 53.5 (45.9–59.3) |
| Range | 18.5–73.1 |
| Sex | |
| Female | 268 (36.4) |
| Male | 469 (63.6) |
| Most common underlying liver disease | |
| Chronic hepatitis C | 174 (23.6) |
| Alcoholic liver disease | 156 (21.2) |
| Hepatocellular carcinoma | 132 (17.9 ) |
| Primary sclerosing cholangitis | 130 (17.6 ) |
| Donor type | |
| Living | 56 (7.6) |
| Deceased | 681 (92.4) |
| Induction immunosuppressiona | |
| Methylprednisolone | 725 (98.4) |
| Daclizumab | 51 (6.9) |
| Muromonab-CD3 (OKT3) | 7 (0.95) |
| Antithymocyte globulin | 2 (0.27) |
| Transplantation with other organ | 49 (6.6) |
| Kidney | 42 (5.7) |
| Heart | 4 (0.5) |
| Kidney and heart | 3 (0.4) |
Abbreviations: IQR, interquartile range; SD, standard deviation.
Data are No. (%) of patients, unless otherwise indicated.
Numbers do not add up to 100%, because some patients used multiple induction immunosuppressant.
CMV Disease After Liver Transplantation
Ninety-two of 737 patients (7.1%, 10.9%, 12.3%, and 12.5% by 3, 6, 12, and 24 months, respectively) developed CMV disease (Table 2). The median time to onset of CMV disease was 69.5 days (range, 5–2442 days) after liver transplantation. The median follow-up time was 2020 days (range, 0–4526 days). The overall incidence of CMV disease was significantly higher (12.1%, 24.1%, and 28.9% vs 7.2%, 8.7%, and 9.5% by 3, 6, and 12 months, respectively; P < .001) in CMV D+/R− than in CMV R+ liver transplant recipients. Only 3 of the 117 CMV D−/R− patients developed CMV disease after liver transplantation (0.9%, 1.8%, and 1.8% by 3, 6, and 12 months, respectively).
Table 2.
Clinical Presentation of Cytomegalovirus (CMV) Disease in a Cohort of Liver Transplant Recipients
| Transplant Recipients |
||||
| Variable | All (n = 737a) | R+ (n = 431) | D+R− (n = 160) | D−R− (n = 117) |
| CMV disease, No. of patients | 92 | 40 | 49 | 3 |
| Incidence, % | ||||
| 3 mo | 7.1 | 7.2 | 12.1 | 0.9 |
| 6 mo | 10.9 | 8.7 | 24.1 | 1.8 |
| 12 mo | 12.3 | 9.5 | 28.9 | 1.8 |
| Time from transplant to onset of CMV disease, median (range), d | 69.5 (5–2442) | 49.5 (5–405) | 127 (18–2442) | 79.5 (45–114) |
| Follow-up time after transplant, median (range), d | 2020 (0–4526) | 2052 (0–4526) | 1927.5 (0–4386) | 1760 (3–4422) |
| CMV disease type, No. of patients | ||||
| Syndrome | 51 | 23 | 26 | 2 |
| Tissue-invasive diseaseb | 41 | 17 | 23 | 1 |
| Upper GI tract | 14 | 8 | 6 | 0 |
| Lower GI tract | 16 | 4 | 11 | 1 |
| Liver | 12 | 3 | 9 | 0 |
| Lung | 5 | 3 | 2 | 0 |
| Other | 2 | 2 | 0 | 0 |
Abbreviations: D−R−, donor and recipient CMV negative; D+R−, donor CMV positive, recipient negative; GI, gastrointestinal; R+, recipient CMV positive.
Totals in R+, D+R−, and D−R− groups do not add up to the overall total because of missing values for CMV serologic status.
Because some patient had multiple organ involvement, the sum of specific tissue invasive disorders exceeds the total.
CMV disease was categorized as CMV syndrome in 51 patients (55.4%) and tissue-invasive CMV disease in 41 patients (44.6 %). Thirty-seven of the 41 patients had definite (biopsy-proven) tissue-invasive disease; only 4 had probable CMV colitis with or without CMV hepatitis. The distribution of tissue involvement for all cases is presented in Table 2.
Association Between TLR2 R753Q SNP and CMV Disease
CMV disease occurred in 11 (8.1%, 20.6%, and 22.8% by 3, 6, and 12 months, respectively) of 52 liver transplant recipients who were TLR2 R753Q homozygotes, 3 (3.6%, 3.6%, and 3.6% by 3, 6, and 12 months, respectively) of 29 who were heterozygotes, and 75 (7.4%, 10.6%, and 12.1% by 3, 6, and 12 months, respectively) of 628 patients who did not have the TLR2 R753Q SNP. Survival analysis using Kaplan–Meier estimation demonstrated a significant association between all CMV disease cases and homozygosity (compared with wild type and heterozygosity) for TLR2 R753Q SNP (P = .044) (Figure 1A). Further analysis demonstrated a statistically significant association between tissue-invasive CMV disease and homozygosity for TLR2 R753Q SNP (P = .001) (Figure 1B).
Figure 1.
Association between Toll-like receptor 2 (TLR2) R753Q SNP homozygosity and cytomegalovirus (CMV) disease–free survival. A, Association of TLR2 R753Q single-nucleotide polymorphism (SNP) homozygosity with CMV free-survival in 737 liver transplant recipients. B, Association of TLR2 R753Q SNP homozygosity with tissue-invasive CMV disease–free survival in 737 liver transplant recipients. C, Association of TLR2 R753Q SNP homozygosity with CMV disease–free survival in CMV-seropositive liver transplant recipients. D, Association of TLR2 R753Q SNP homozygosity with CMV disease–free survival in CMV D+R− mismatch patients.
In a subgroup analysis aimed to control for CMV serologic status and to determine associations between TLR2 R753Q SNP and primary or reactivation CMV disease, there was a statistically significant association between homozygosity (compared with wild type and heterozygosity) for TLR2 R753Q SNP and reactivation of CMV disease in CMV R+ liver transplant recipients (P = .034) (Figure 1C). However, this significant association was not observed for primary CMV disease in CMV D+/R− liver transplant recipients (P = .67) (Figure 1D).
Univariate Cox proportional hazard models among CMV R+ and D+/R− patients are summarized in Table 3. CMV R+ patients had a significantly lower risk of any type of CMV disease, compared with D+/R− patients (hazard ratio [HR], 0.29; 95% CI, .19–.43; P < .0001). Both acute rejection (HR, 3.65; 95% CI, 2.24–5.94; P < .0001) and bacterial infections (HR, 3.07; 95% CI, 1.77–5.34; P < .0001) were associated with an increased risk of CMV disease. TLR2 R753Q homozygosity trended toward an increased risk of CMV disease (HR, 1.84; 95% CI, 0.98–3.47; P = .059). No specific immunosuppression at 30 days after transplantation was associated with CMV disease. For the outcome of tissue-invasive CMV disease, TLR2 R753Q homozygosity (HR, 3.22; 95% CI, 1.48–7.00; P = .003), acute rejection (HR, 5.98; 95% CI, 2.91–12.29; P < .0001), and bacterial infection (HR, 3.86; 95% CI, 1.73–8.62; P = .001) were significantly associated with an increased risk. In contrast, CMV R+ (compared with D+/R−) patients (HR, 0.27; 95% CI, .15–.51; P < .0001) and older patients (10-year HR, 0.71; 95% CI, .55–.93; P = .0127) had a lower risk of tissue-invasive CMV disease. Specific immunosuppressive drugs were not significantly associated with tissue-invasive CMV disease.
Table 3.
Univariate Cox Proportional Hazard Models in Cytomegalovirus (CMV) R+ and D+R− Patients Assessing Associations With All Types and Tissue-Invasive Form of CMV Disease
| All Types of CMV Disease |
Tissue-Invasive CMV Disease |
|||
| Variable | Hazard Ratio (95% CI) | P | Hazard Ratio (95% CI) | P |
| CMV R+ vs D+/R− | 0.29 (.19–.43) | <.0001 | 0.27 (.15–.51) | <.0001 |
| TLR2 R753Q homozygosity | 1.84 (.98–3.47) | .0590 | 3.22 (1.48–7.00) | .0031 |
| Age (by decade) | 0.84 (.69–1.01) | .0651 | 0.71 (.55–.93) | .0127 |
| Acute allograft rejectiona | 3.65 (2.24–5.94) | <.0001 | 5.98 (2.91–12.29) | <.0001 |
| Bacterial infectiona | 3.07 (1.77–5.34) | <.0001 | 3.86 (1.73–8.62) | .0010 |
| Chronic hepatitis C | 0.955 (.585–1.557) | .8531 | 0.651 (.288–1.473) | .3031 |
| Immunosuppressive therapy at 30 d | ||||
| Mycophenolate mofetil | 1.05 (.65–1.70) | .8471 | 2.01 (.84–4.79) | .1145 |
| Tacrolimus | 0.65 (.38–1.12) | .1198 | 0.84 (.35–2.00) | .6903 |
| Sirolimus | 0.87 (.12–6.26) | .8913 | 2.10 (.29–15.28) | .4631 |
| Azathioprine | 1.16 (.68–1.97) | .5861 | 0.68 (.27–1.73) | .4141 |
| Cyclosporine | 0.93 (.47–1.86) | .8483 | 0.92 (.33–2.59) | .8782 |
| Prednisone | 0.75 (.11–5.42) | .7793 | 0.28 (.04–2.03) | .2070 |
Abbreviations: CI, confidence interval; D+/R−, donor CMV positive, recipient CMV negative; R+, recipient CMV seropositive; TLR2, toll-like receptor 2.
All allograft rejections and bacterial infections during the follow-up period were accounted for using time-dependent predictors in the Cox proportional hazard model for possible influence of 90- and 30-day periods, respectively.
In a multivariable Cox proportional hazard model among CMV D+/R− and R+ patients that accounted for patient age, bacterial infection, acute allograft rejection, and CMV serologic status, a marginal association was observed between homozygosity (compared with wild type and heterozygosity) for TLR2 R753Q SNP and a higher risk of any CMV disease (HR, 1.85; 95% CI, 0.97–3.528]; P = .062) (Table 4). Being CMV R+ was associated with a significantly lower risk (HR, 0.280; 95% CI, .18–.435; P < .0001), whereas having had bacterial infection (HR, 3.033; 95% CI, 1.70–5.411; P = .002) or an acute rejection (HR, 3.531; 95% CI, 2.113–5.899; P < .0001) were associated with significantly higher risk of CMV disease. A similar multivariable analysis of tissue-invasive CMV disease among D+/R− and R+ patients demonstrated that being a homozygote for TLR2 R753Q SNP was significantly associated with a higher risk (HR, 3.407; 95% CI, 1.518–7.644; P = .0029). Likewise, this multivariate analysis demonstrated a significant association between tissue-invasive CMV disease and CMV D+/R− serostatus, acute rejection, and bacterial infections after liver transplantation (Table 4).
Table 4.
Multivariable Cox Proportional Hazard Models in Cytomegalovirus (CMV) R+ and D+/R− Patients Assessing Associations Between CMV Status, TLR2 753Q Single-Nucleotide Polymorphism Homozygosity, Patient Age, Acute Rejection, and Bacterial Infection With All Types and Tissue-Invasive Form of CMV Disease
| All Types of CMV Disease |
Tissue-Invasive CMV Disease |
|||
| Hazard Ratio (95% CI) | P | Hazard Ratio (95% CI) | P | |
| CMV R+ vs D+/R− | 0.28 (.18–.44) | <.0001 | 0.30 (.15–.58) | .0002 |
| TLR2 R753Q homozygosity | 1.85 (.97–3.53) | .0620 | 3.41 (1.52–7.64) | .0029 |
| Age (by decade) | 1.03 (.85–1.26) | .7325 | 0.97 (.74–1.28) | .8322 |
| Acute allograft rejectiona | 3.53 (2.11–5.90) | <.0001 | 5.25 (2.45–11.28) | <.0001 |
| Bacterial infectiona | 3.03 (1.70–5.41) | <.0002 | 3.23 (1.40–7.45) | .0059 |
Abbreviation: CI, confidence interval; D+/R−, donor CMV positive, recipient CMV negative; R+, recipient CMV seropositive; TLR2, toll-like receptor 2.
All allograft rejections and bacterial infections during the follow-up period were accounted for using time-dependent predictors in the Cox proportional hazard model for possible influence of 90- and 30-d time periods, respectively.
DISCUSSION
This is the largest study, to our knowledge, to demonstrate that homozygosity for the TLR2 R753Q SNP was significantly associated with a higher risk of CMV disease after liver transplantation. This seems to be independent of traditional risk factors, such as CMV serologic status, bacterial infections, and acute rejection. The association was most significant with tissue-invasive CMV disease.
TLR2 is a member of a family of pattern recognition receptors for which the main function is to serve as initial sensors of pathogen-associated molecular patterns in the environment [22]. Expressed on the cell surface, TLR2 is a pleiotrophic receptor that signals to the host of the presence of a wide array of nonself molecular patterns, including peptidoglycan and lipopeptides from gram-positive bacteria [23, 24] and envelope glycoproteins B and H of CMV [9, 12, 13], among others. Recognition of glycoproteins B and H of CMV by TLR2 initiates a series of intracellular signaling events mediated by the adapter protein MyD88 (myeloid differentiation D protein 88) and transcription factor nuclear factor (NF-κB), culminating in the release of antiviral peptides, chemokines, and cytokines that subsequently prime the adaptive immune response [9, 12, 13]. The data presented in this clinical study suggest the importance of the TLR2 signaling pathway in the pathogenesis and control of CMV disease in humans.
TLR2 is encoded by the tlr2 gene, which maps on chromosome 4q32 [25, 26]. The tlr2 gene has been reported to contain ≥89 SNPs in its promoter, intron, and exon regions [26]. Of these SNPs, TLR2 R753Q has been investigated the most because of its functional consequence (ie, amino acid substitution in the expressed protein) and its prevalence in the population [9, 11, 20, 24–28]. Depending on the study, the allele frequency of R753Q is reported to range from 2.7% to 12% [9, 11, 20, 24–28]. Studies in vitro have demonstrated that the R753Q SNP impairs the ability of TLR2 to sense its prototypic ligands peptidoglycan and lipopeptides [23, 24, 28]. Likewise, R753Q has abrogated the ability of cells to recognize glycoprotein B of CMV in vitro [9]. In contrast to cells expressing the wild-type TLR2 gene, human embryonic kidney 293 cells that possess the mutant TLR2 R753Q gene had a markedly impaired ability to respond to stimulation with the envelope glycoprotein B of human CMV, as evidenced by the lack of NF-κB activation and interleukin-8 secretion [9].
Innate immune gene polymorphisms including TLR SNPs are being studied extensively for their association with infectious diseases on the basis of a hypothesis that, given the position of the innate immunity genes and the proteins that they encode at the interface of the host and environment, even minor variation in these genes could have a major impact on downstream responses that could be critical for host defense or disease pathogenesis [29]. For CMV disease in humans, the relevance of TLR2 R753Q SNP was initially suggested by our prior report, in which we described the significant association between the presence of this mutation with a higher level of CMV replication (ie, viral load) and a higher incidence of CMV disease [9, 11]. In this pilot study, homozygosity for TLR2 R753Q was marginally associated with CMV disease in a cohort of 92 HCV-infected liver transplant recipients [11]. In addition, there was a higher level of virus in patients who were homozygous for TLR2 R753Q SNP [11]. However, the multivariate analysis demonstrated that the association was, at best, marginal, and it did not reach statistical significance [11]. Moreover, the study was conducted in a relatively modest and homogenous population of patients with chronic HCV infection [11], and thus, it would need validation in an independent cohort. Because there were compelling in vitro and animal data, we aimed to confirm or refute the original observation by performing the current study.
This larger study, which included >700 patients who received transplants for various indications, confirmed our previous report [11] that, indeed, homozygosity for TLR2 R753Q SNP was significantly associated with a higher incidence of CMV disease, especially reactivation disease and tissue-invasive CMV disease, after liver transplantation. The association remained strong even in the multivariable model, after accounting for well-known risk factors, such as CMV serologic status, bacterial infections, and acute allograft rejection [1, 3, 17, 19]. Moreover, HCV infection as the underlying indication for liver transplantation did not influence this association. The current study, together with prior clinical reports and several in vitro and experimental investigations [9, 11–14], implicates the importance of this receptor and its signaling pathway in the pathogenesis and clinical course of CMV disease after liver transplantation. These reports implicate intrinsic genetic factors in infectious disease predisposition and clinical course. Although previous studies identifying risk factors for CMV disease after transplantation have focused mainly on patient-related clinical variables [30, 31], the field should also now focus on investigating genetic markers and incorporating them with clinical risk factors in defining better strategies for prevention and treatment.
In conclusion, this study found a significant association between TLR2 R753Q SNP and a higher incidence of CMV disease. Statistical significance was stronger when homozygosity of the TLR2 R753Q SNP was examined in association with the more severe form of CMV disease (ie, tissue-invasive form). These observations should encourage further studies to identify other genetic markers for CMV disease to better identify transplant recipients at high risk.
Notes
Acknowledgments.
We thank Ms Teresa Hoff for her secretarial assistance in the preparation of this manuscript.
Financial support.
This work was supported by National Institutes of Health, National Center for Research Resources, Center for Translational Science Activities (NCRR CTSA) (grant UL1 RR024150); the CR20 Award from the Clinical and Translational Science Activities, Mayo Clinic (to R. R. R.); and the Mayo Transplant Center Scholarly Award (to R. R. R). The contents of this article are solely the responsibility of the authors and do not necessarily represent the official views of the National Institutes of Health.
Potential conflicts of interest.
All authors: No reported conflicts.
All authors have submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest. Conflicts that the editors consider relevant to the content of the manuscript have been disclosed.
References
- 1.Razonable RR. Cytomegalovirus infection after liver transplantation. Liver Transpl. 2010;16:S45–53. [Google Scholar]
- 2.Asberg A, Humar A, Rollag H, et al. Oral valganciclovir is noninferior to intravenous ganciclovir for the treatment of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant. 2007;7:2106–13. doi: 10.1111/j.1600-6143.2007.01910.x. [DOI] [PubMed] [Google Scholar]
- 3.Humar A, Lebranchu Y, Vincenti F, et al. The efficacy and safety of 200 days valganciclovir cytomegalovirus prophylaxis in high-risk kidney transplant recipients. Am J Transplant. 2010;10:1228–37. doi: 10.1111/j.1600-6143.2010.03074.x. [DOI] [PubMed] [Google Scholar]
- 4.Paya C, Humar A, Dominguez E, et al. Efficacy and safety of valganciclovir vs. oral ganciclovir for prevention of cytomegalovirus disease in solid organ transplant recipients. Am J Transplant. 2004;4:611–20. doi: 10.1111/j.1600-6143.2004.00382.x. [DOI] [PubMed] [Google Scholar]
- 5.Paya CV, Wilson JA, Espy MJ, et al. Preemptive use of oral ganciclovir to prevent cytomegalovirus infection in liver transplant patients: a randomized, placebo-controlled trial. J Infect Dis. 2002;185:854–60. doi: 10.1086/339449. [DOI] [PubMed] [Google Scholar]
- 6.Cummins NW, Deziel PJ, Abraham RS, Razonable RR. Deficiency of cytomegalovirus (CMV)-specific CD8+ T cells in patients presenting with late-onset CMV disease several years after transplantation. Transpl Infect Dis. 2009;11:20–7. doi: 10.1111/j.1399-3062.2008.00344.x. [DOI] [PubMed] [Google Scholar]
- 7.Eid AJ, Brown RA, Arthurs SK, et al. A prospective longitudinal analysis of cytomegalovirus (CMV)-specific CD4+ and CD8+ T cells in kidney allograft recipients at risk of CMV infection. Transpl Int. 2010;23:506–13. doi: 10.1111/j.1432-2277.2009.01017.x. [DOI] [PubMed] [Google Scholar]
- 8.Eid AJ, Razonable RR. New developments in the management of cytomegalovirus infection after solid organ transplantation. Drugs. 2010;70:965–81. doi: 10.2165/10898540-000000000-00000. [DOI] [PubMed] [Google Scholar]
- 9.Brown RA, Gralewski JH, Razonable RR. The R753Q polymorphism abrogates toll-like receptor 2 signaling in response to human cytomegalovirus. Clin Infect Dis. 2009;49:e96–9. doi: 10.1086/644501. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Cervera C, Lozano F, Linares L, et al. Influence of mannose-binding lectin gene polymorphisms on the invasiveness of cytomegalovirus disease after solid organ transplantation. Transplant Proc. 2009;41:2259–61. doi: 10.1016/j.transproceed.2009.06.056. [DOI] [PubMed] [Google Scholar]
- 11.Kijpittayarit S, Eid AJ, Brown RA, Paya CV, Razonable RR. Relationship between Toll-like receptor 2 polymorphism and cytomegalovirus disease after liver transplantation. Clin Infect Dis. 2007;44:1315–20. doi: 10.1086/514339. [DOI] [PubMed] [Google Scholar]
- 12.Boehme KW, Guerrero M, Compton T. Human cytomegalovirus envelope glycoproteins B and H are necessary for TLR2 activation in permissive cells. J Immunol. 2006;177:7094–102. doi: 10.4049/jimmunol.177.10.7094. [DOI] [PubMed] [Google Scholar]
- 13.Compton T, Kurt-Jones EA, Boehme KW, et al. Human cytomegalovirus activates inflammatory cytokine responses via CD14 and Toll-like receptor 2. J Virol. 2003;77:4588–96. doi: 10.1128/JVI.77.8.4588-4596.2003. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Szomolanyi-Tsuda E, Liang X, Welsh RM, Kurt-Jones EA, Finberg RW. Role for TLR2 in NK cell-mediated control of murine cytomegalovirus in vivo. J Virol. 2006;80:4286–91. doi: 10.1128/JVI.80.9.4286-4291.2006. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Humar A, Michaels M. American Society of Transplantation recommendations for screening, monitoring and reporting of infectious complications in immunosuppression trials in recipients of organ transplantation. Am J Transplant. 2006;6:262–74. doi: 10.1111/j.1600-6143.2005.01207.x. [DOI] [PubMed] [Google Scholar]
- 16.Ljungman P, Griffiths P, Paya C. Definitions of cytomegalovirus infection and disease in transplant recipients. Clin Infect Dis. 2002;34:1094–7. doi: 10.1086/339329. [DOI] [PubMed] [Google Scholar]
- 17.Arthurs SK, Eid AJ, Pedersen RA, et al. Delayed-onset primary cytomegalovirus disease after liver transplantation. Liver Transpl. 2007;13:1703–9. doi: 10.1002/lt.21280. [DOI] [PubMed] [Google Scholar]
- 18.Razonable RR, van Cruijsen H, Brown RA, et al. Dynamics of cytomegalovirus replication during preemptive therapy with oral ganciclovir. J Infect Dis. 2003;187:1801–8. doi: 10.1086/375194. [DOI] [PubMed] [Google Scholar]
- 19.Razonable RR, Rivero A, Rodriguez A, et al. Allograft rejection predicts the occurrence of late-onset cytomegalovirus (CMV) disease among CMV-mismatched solid organ transplant patients receiving prophylaxis with oral ganciclovir. J Infect Dis. 2001;184:1461–4. doi: 10.1086/324516. [DOI] [PubMed] [Google Scholar]
- 20.Eid AJ, Brown RA, Paya CV, Razonable RR. Association between toll-like receptor polymorphisms and the outcome of liver transplantation for chronic hepatitis C virus. Transplantation. 2007;84:511–6. doi: 10.1097/01.tp.0000276960.35313.bf. [DOI] [PubMed] [Google Scholar]
- 21.Hamann L, Hamprecht A, Gomma A, Schumann RR. Rapid and inexpensive real-time PCR for genotyping functional polymorphisms within the Toll-like receptor -2, -4, and -9 genes. J Immunol Methods. 2004;285:281–91. doi: 10.1016/j.jim.2003.12.005. [DOI] [PubMed] [Google Scholar]
- 22.Medzhitov R, Preston-Hurlburt P, Janeway CA., Jr A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature. 1997;388:394–7. doi: 10.1038/41131. [DOI] [PubMed] [Google Scholar]
- 23.El-Helou O, Berbari EF, Brown RA, Gralewski JH, Osmon DR, Razonable RR. Functional assessment of Toll-like receptor 2 and its relevance in patients with Staphylococcus aureus infection of joint prosthesis. Hum Immunol. 2011;72:47–53. doi: 10.1016/j.humimm.2010.10.001. [DOI] [PubMed] [Google Scholar]
- 24.Lorenz E, Mira JP, Cornish KL, Arbour NC, Schwartz DA. A novel polymorphism in the toll-like receptor 2 gene and its potential association with staphylococcal infection. Infect Immun. 2000;68:6398–401. doi: 10.1128/iai.68.11.6398-6401.2000. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.Schwartz DA, Cook DN. Polymorphisms of the Toll-like receptors and human disease. Clin Infect Dis. 2005;41(Suppl 7):S403–7. doi: 10.1086/431985. [DOI] [PubMed] [Google Scholar]
- 26.Texereau J, Chiche JD, Taylor W, Choukroun G, Comba B, Mira JP. The importance of Toll-like receptor 2 polymorphisms in severe infections. Clin Infect Dis. 2005;41(Suppl 7):S408–15. doi: 10.1086/431990. [DOI] [PubMed] [Google Scholar]
- 27.Brown RA, Gralewski JH, Eid AJ, Knoll BM, Finberg RW, Razonable RR. R753Q single-nucleotide polymorphism impairs toll-like receptor 2 recognition of hepatitis C virus core and nonstructural 3 proteins. Transplantation. 2010;89:811–5. doi: 10.1097/TP.0b013e3181cbac18. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Schroder NW, Hermann C, Hamann L, Gobel UB, Hartung T, Schumann RR. High frequency of polymorphism Arg753Gln of the Toll-like receptor-2 gene detected by a novel allele-specific PCR. J Mol Med. 2003;81:368–72. doi: 10.1007/s00109-003-0443-x. [DOI] [PubMed] [Google Scholar]
- 29.Lazarus R, Vercelli D, Palmer LJ, et al. Single nucleotide polymorphisms in innate immunity genes: abundant variation and potential role in complex human disease. Immunol Rev. 2002;190:9–25. doi: 10.1034/j.1600-065x.2002.19002.x. [DOI] [PubMed] [Google Scholar]
- 30.Eid AJ, Arthurs SK, Deziel PJ, Wilhelm MP, Razonable RR. Clinical predictors of relapse after treatment of primary gastrointestinal cytomegalovirus disease in solid organ transplant recipients. Am J Transplant. 2010;10:157–61. doi: 10.1111/j.1600-6143.2009.02861.x. [DOI] [PubMed] [Google Scholar]
- 31.Limaye AP, Bakthavatsalam R, Kim HW, et al. Late-onset cytomegalovirus disease in liver transplant recipients despite antiviral prophylaxis. Transplantation. 2004;78:1390–6. doi: 10.1097/01.tp.0000145989.22373.03. [DOI] [PubMed] [Google Scholar]