A non-interventional study of the genetic polymorphisms of NOD2 associated with increased mortality in non-alcoholic liver transplant patients

Immunosuppression, as in liver transplant patients, carries a high risk of life threatening infections such as pneumonia or blood stream infections (BSI). Because calcineurin inhibitors are used as immunosuppressive agents to avoid organ rejection, infections have emerged in the last 20 years as the main cause of death in the first year after transplantation [1, 2].

Several risk factors for infections after liver transplantation (LTX) have been identified, such as being male sex, having a prolonged cold ischemia time, acute liver failure, rejection treatment, relaparotomy due to bleeding or bile, and retransplantation within 30 days [3‐5]. Recent reports indicate that single nucleotide polymorphisms (SNPs) are involved in a variety of diseases. Mutations of the toll-like receptor 4 (TLR-4) genes are associated with an increased incidence of inflammatory bowel disease due to bowl alterations such as changes in the bacterial load and translocation [6], whereas TLR-4 mutations in bone-marrow transplant patients are associated with an increased risk for graft-versus-host disease (GvHD) [7]. Nucleotide oligomerisation domain 2 (NOD2) polymorphisms, as a part of the innate immune system, have been shown to be protective against GvHD [7]. Several SNPs in specific genes of liver patients have been evaluated, with conflicting results. Mannose binding lectin (MBL) in the donor was found to carry a higher risk for postoperative life threatening infections [8, 9]. Another clinical study indicates that an MBL gene polymorphism was associated with an increased risk for the development of hepato-cellular carcinoma in chronic hepatitis C patients [10]. The TLR 2 and TLR4-receptors are assumed to recognize hepatitis C virus. The association between TLR polymorphisms and outcome after transplantation due to chronic hepatitis C infection was evaluated in a clinical study [11]. The authors found that a homozygous TLR 2 polymorphism was associated with a higher incidence of poor graft function and higher mortality.

NOD2 gene polymorphism is associated with an increased incidence of liver and intestine failure in patients following combined liver and small bowl transplantation [12].

Recently, a group from Rotterdam [13] evaluated 50 polymorphisms in 181 liver transplant patients and their corresponding donors with respect to organ rejection and infections in the recipient. They found no correlation between the gene variants and bacterial and/or fungal infections.

The aim of our study was to evaluate the impact of 13 different SNPs, including in NOD2 and TLR 4, on transplant-associated complications such as infection, need for dialysis and the outcomes of the recipients.

During the observation period of 2/2009 to 1/2012, a total of 231 patients were recruited in this prospective, non-interventional study. All patients received liver transplantations at the Medical Center University Essen, Department of General -, Visceral- and Transplant Surgery.

All patients were transplanted with whole liver from a brain-dead donor.

Exclusion criteria were recipient age ≤ 18 years and lack of a blood sample from the recipient.

This study was conducted in accordance with the amended Declaration of Helsinki. Local institutional review boards of the University Essen approved the protocol, and written informed consent was obtained from all patients.

Our study indicates that wild type NOD2 confers a survival benefit to non-alcoholic patients who receive a liver transplant. We recorded a 36% higher survival in patients with the wild type gene variants compared to the NOD 2 mutation gene variants in this cohort. However, these results were not reproducible for patients with alcoholic disease. In contrast, in patients with alcoholic disease, the gene variant of NOD2 seems to increase survival, though this finding was not statistically significant. Patients with alcoholic disease were significantly older according to statistical analysis, but the difference of 3 years (55 vs. 52 years) seems clinically negligible.

EAD is a serious complication after liver transplantation, which is associated with a higher retransplantation rate and higher mortality [19‐21]. EAD was almost 2 times more common in patients with non-alcoholic disease compared to alcoholic patients who received a liver transplant. However, the protective effect of the NOD2 gene with wild type variants overruled the EAD effect.

Recent reports have provided evidence that NOD2 gene variants influence the severity of graft-versus-host-disease (GVHD) and the transplant survival in bone marrow transplant (BMT) patients.

The Elmaagacli group demonstrated a significantly higher incidence of GVHD in patients with gene variants of NOD2 following BMT compared with patients who had wild type NOD2 [7]. These data are in agreement with a study by Holler et al. [22] in which there was an increased risk of GVHD in patients with an NOD2 gene variant. The authors also reported an increased risk of mortality with the gene variant, which is in contrast with the findings of the Elmaagacli group [7].

The Pittsburgh group evaluated the NOD 2 gene variant in simultaneous liver and small bowel transplanted pediatric patients [12]. They reported that NOD2 gene variants were associated with increased liver and intestinal failure in patients with short-gut syndrome.

Although NOD2, as a part of the innate immune system, plays a key role in preventing bacterial infections, we did not find a difference among patients with and without CSI. Moreover, NOD2 mutations were not associated with a decreased incidence of dialysis use; wild type NOD2 in non-alcoholic patients may have an independent protective effect for survival.

More than a quarter of our patients experienced CSI, which was associated with significantly higher rates of dialysis use, longer ventilation time, length of ICU – and hospital stay, and higher mortality. In an earlier study conducted by our department, the rate of CSI (pneumonia and sepsis) was 32.3% (24% sepsis rate and 8.3% pneumonia rate) [2]. The reason for the higher CSI rate may be related to a different patient population. While only patients with a postmortem organ were recruited in the current study, 24% of patients in the previous study were transplanted with a graft from a living donor. Biliary complications, including bile leaks, which are associated with sepsis, range from 0.3 - 22% in whole organ transplantation [23, 24], which is significantly lower than with split liver transplantation with a living donor graft. Some groups reported biliary complications in split liver transplantation of up to 60% [25], which may contribute to a lower sepsis rate in the current study despite a higher MELD score compared with our own data in the last decade.

A recently published study [26] evaluated the risk factors for infections in 367 patients. The authors reported a CSI (pneumonia and blood stream infection) rate of 28.8%, which is similar to the incidence in our study. In line with this study, Sganga et al. [27] identified a blood stream infection rate of 28%. Unfortunately, neither study reported the survival rate in patients with infection. Our study indicated a nearly 50% decrease in the one-year-survival for patients who develop a CSI, which is significantly higher than in other groups. Sun et al. [28] evaluated the risk factors for infections in the MELD era. The group stated that the recipient age (OR =1.09) and the MELD score affect infection. They reported an overall (patients with and without infections) one-year survival rate of 76.7% in the Pre-MELD era and 82.5% in the MELD era. In our study, the one-year survival rate was 72.3% for all patients. The MELD scores in both studies were comparable, but there is a difference of 10% in the one-year survival of all patients, which may be related to the quality of the donor graft. While in the UNOS region, only 32% of the transplanted grafts have a DRI of > 1.5; this rate accounts for 65% in the EUROTRANSPLANT region. Moreover, only 6% of the transplanted organs in the UNOS region have a DRI > 2, but 23% in the EUROTRANSPLANT area have a DRI > 2, which is associated with a higher graft failure and lower patient survival [29]. This may in part explain the higher mortality.

There are several studies reporting a worse outcome of liver transplantation in patients with kidney dysfunction and dialysis [30‐32]. The registry data of liver transplant patients with postoperative kidney dysfunction demonstrate a significantly worse 2-year survival compared with patients with adequate or mild kidney dysfunction (55% vs. 76%, p < 0.05) [33]. These data are in line with a retrospective study that evaluated the risk factors in terms of the survival rate after liver transplantation [34]. Patients with dialysis-dependent kidney failure had a one-year survival of less than 50%.

These data are comparable with our study. Sixty-six percent of patients with CSI had dialysis-dependent kidney failure, which was associated with a one-year survival 64.7%. Sun et al. reported of a dialysis-dependent kidney failure in less than 10% of their transplanted patients. In contrast to that study, 32.3% of our patients required dialysis treatment due to kidney failure, which affects their outcomes. In this context, it is indispensible to highlight the significantly higher dialysis rate in patients with non-alcoholic disease than in alcoholic patients in our cohort. Despite this fact, wild type NOD2 was protective in this patient cohort with respect to their long-term survival.

Our study has some limitations. First, it was a single-center study. Because we conducted an observational study, there is no real control group. We did not stratify or evaluate the patients’ outcomes with respect to the transplant surgeon or anesthesia team. However because 2007 we have had a constant dedicated surgery and anesthesia team who take care for these patients.

In conclusion, our data indicates wild type NOD2 offers a survival benefit in liver transplant patients with non-alcoholic disease. Patients with the NOD2 gene variant should have closer follow-up as part of the standard of care. The other SNPs that we tested did not significantly influence the CSI and survival rates in this cohort. Transplant-related factors may be more dominant risk factors for these patients than the genetic polymorphisms.