The regulatory toll-like receptor 4 genetic polymorphism rs11536889 is associated with renal, coagulation and hepatic organ failure in sepsis patients

Adult Caucasian patients admitted to the University Medical Center Goettingen (UMG) intensive care units (ICUs) between April 2012 and May 2013 were screened daily according to the American College of Chest Physicians/Society of Critical Care Medicine (ACCP/SCCM) criteria for sepsis, severe sepsis, or septic shock[29, 30]. Caucasian origin was assessed by questioning the patients, their next of kin or their legal representatives. The patient exclusion criteria were as follows: 1. age younger than 18 years; 2. pregnancy, nursing an infant, or planning to become pregnant or nurse an infant; 3. receiving an immunosuppressive therapy (e.g., cyclosporine or azathioprine) or cancer-related chemotherapy; 4. a documented or suspected acute myocardial infarction within the previous six weeks; 5. a history of New York Heart Association functional class IV chronic heart failure: 6. human immunodeficiency virus infection or end-stage process (e.g., progressive multifocal leukoencephalopathy or systemic Mycobacterium avium infection); 7. morbidity and death were considered imminent, the patient was classified as “do not resuscitate” or “do not treat”, or the patient and/or a legally authorized representative was not committed to aggressive management; 8. the patient was not expected to survive the observation period of 28 days or was not likely to be given life support because of a preexisting, uncorrectable medical condition, including a poorly controlled neoplasm, end-stage lung disease, or home oxygen requirement; 9. the patient was in a chronic vegetative state or had a similar long-term neurologic condition; 10. participation in any other investigational study (drug or device); 11. the patient was unwilling or unable to be fully evaluated during the study period; and 12. the patient was a study-site employee or was an immediate family member of a study-site employee involved in the study. The study was approved by the University of Goettingen ethics committee, Goettingen, Germany (15/1/12) and conformed to the Declaration of Helsinki ethical principles (Seoul, 2008). Written informed consent was obtained either from patients or their legal representatives.

The Sequential Organ Failure Assessment (SOFA)[31] and Acute Physiology and Chronic Health Evaluation (APACHE) II[32] scores were evaluated at the onset of sepsis. Organ function was assessed subsequently on days 2, 3, 5, 7, 14, 21 and 28, and organ failure was quantified, with the SOFA score as the primary outcome variable. Patients were followed up for a maximum of 90 days, and their deaths were recorded as a secondary outcome variable. The necessity of mechanical ventilation, vasopressor administration, or renal-replacement therapy as well as the ICU duration was recorded as secondary variables.

Peripheral blood monocytes (PBMCs) from approximately 30 ml of heparinized peripheral blood were isolated through Ficoll density gradient centrifugation according to standard procedures described previously[33]. Cell preparations were routinely assessed for viability (>95%) by trypan blue dye exclusion.

Genomic DNA (gDNA) was purified from PBMCs using the AllPrep DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The isolated nucleic acid concentration and purity were determined by 260 and 280 nm optical density readings. DNA integrity was evaluated through 0.6% agarose gel electrophoresis.

Genotyping was performed using 4 ng of PBMC-derived gDNA in a commercially available genotyping assay (Assay ID C_31784034_10, Applied Biosystems, Darmstadt, Germany) in a total volume of 10 μl. The reactions were performed in a StepOnePlus sequence detection system (Applied Biosystems, Darmstadt, Germany) according to the supplier’s instructions.

The Hardy-Weinberg equilibrium exact test for deviation was performed using an online calculator, which was provided by the Institute of Human Genetics, Helmholtz Center Munich, Germany (http://​ihg.​gsf.​de/​cgi-bin/​hw/​hwa1.​pl). Statistical analyses were performed with the Statistica (StatSoft, Tulsa, Oklahoma, USA, version 10) or R (The R Foundation for Statistical Computing, version 3.0.0) software. Significance, based on contingency tables, was calculated using two-sided Fisher’s exact or chi-square tests, as appropriate. Two continuous variables were compared using the Mann-Whitney test. To estimate the significance of the clinical observations over the 28-day period, we fitted a linear regression model with the parameters day, genotype, and genotype-day interaction. The results were visualized by calculating the means and 95% confidence intervals (CIs) from normal distributions at each time point. Time-to-event data were compared using the log-rank test from the Statistica package survival. A p-value less than 0.05 was considered significant.

A total of 212 adult Caucasian patients with sepsis were enrolled into this study. Two patients were excluded; one patient fulfilled an exclusion criterion, as a B cell lymphoma diagnosis became known, and the other patient was excluded because informed consent was withdrawn by his legally authorized representative. Subsequently, the study population comprised 210 patients, in which 134 were male (64%), and 76 were female (36%; Table 1). The patient ages ranged from 19 to 91 (median, 65). The sepsis subtypes were sepsis/severe sepsis (n = 100) and septic shock (n = 110). At baseline, the patient disease severity SOFA and APACHE II scores were 8.6 ± 4.1 and 21.3 ± 7.4, respectively (Table1). The comorbidities comprised hypertension, myocardial infarction history, chronic obstructive pulmonary disease (COPD), renal dysfunction, diabetes mellitus, chronic liver diseases, cancer history, and stroke history (Table 1).

TLR4 rs11536889 was successfully genotyped in all subjects. The genotype distribution was 146:62:2 (GG:GC:CC), which was consistent with the Hardy-Weinberg equilibrium (p = 0.12). The resulting 0.16 minor allele frequency was similar to that given for Caucasians in public databases. The rs11536889 GC and CC genotypes were pooled together because the size of the CC genotype group was too small (n = 2). Subsequently, patients of genotype GG were compared to that of CG/CC. There was no difference regarding age, gender, or body mass index related to the TLR4 rs11536889 genotype. A comparison between the G homozygous patients and C allele carriers revealed no significant difference between the proportion of patients with sepsis/severe sepsis and septic shock at baseline (day 1 of sepsis; p = 0.4576). Furthermore, there were no SOFA and APACHE II score differences regarding the TLR4 rs11536889 genotypes at sepsis onset, and there were no significant preexisting conditions differences between the TLR4 rs11536889 genotypes (Table 1). Moreover, the recent surgical histories and primary infection sites showed no significant difference with respect to the genotype distribution (Table 1).

Disease progression was monitored by SOFA score changes during the patient ICU stays. The scores and the need for organ support were recorded on days 1, 2, 3, 5, 7, 14, 21, and 28. Although no differences in disease scores were observed at sepsis onset, the TLR4 rs11536889 GG patients experienced significantly higher SOFA scores over time (p = 0.0005) compared with the C allele carriers (Table 2). The three organ-specific SOFA scores were significantly different between the two groups; the GG patients presented higher SOFA-renal scores (p = 0.0005), SOFA-coagulation scores (p = 0.0245), and SOFA-hepatic scores (p < 0.0001; Table 2, Figure 1). An overall linear model was fitted to the values, which included the various time points. This model also revealed a significant genotype effect; the GG patients presented higher SOFA scores than did the GC/CC subjects (p = 0.015; Figure 2). The mean SOFA scores of the two CC patients were 11.6 ± 3.5 (mean ± SD). Among all SOFA scores, the minimum and maximum were 0 and 23, respectively.

The 28-day and 90-day mortality analyses yielded no significant result between the groups (p = 1.0000 and p = 0.8698, respectively; Table 2 (Additional file1: Figure S1)). Both at the beginning and over the observational period, there was no significant difference between the GG patients and C allele carriers regarding organ support requirement (mechanical ventilation, vasopressor use, and renal replacement therapy; Tables 1 and2). The mean ICU stay duration of the GG survivors did not differ significantly from that of the GC/CC survivors (Table 2). Additionally, there was a significantly higher gram-negative infection incidence rate among the C allele carriers (81%) compared with that in the GG patients (62%; p = 0.0062) (Table 3).