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Human Molecular Genetics Advance Access originally published online on September 14, 2007
Human Molecular Genetics 2007 16(24):3071-3080; doi:10.1093/hmg/ddm265
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Heterozygosity of mannose-binding lectin (MBL2) genotypes predicts advantage (heterosis) in relation to fatal outcome in intensive care patients

Dorthe Hellemann1,2, Anders Larsson6, Hans O. Madsen2, Jan Bonde3, Jens Otto Jarløv4, Jørgen Wiis3, Torsten Faber1, Jørn Wetterslev5 and Peter Garred2,*

1 Department of Anaesthesiology and Intensive Care, Herlev,, 2 Department of Clinical Immunology, sect.7631,, 3 Intensive Care Unit 4131,, 4 Department of Clinical Microbiology, Herlev,, 5 Copenhagen Trial Unit, Copenhagen University Hospital, Rigshospitalet, Denmark and 6 Clinical Institute, Århus University, Denmark

* To whom correspondence should be addressed at: Department of Clinical Immunology, sect. 7631, Copenhagen University Hospital, Rigshospitalet, Blegdamsvej 9, 2100 Copenhagen O, Denmark. Tel: +45 35457631; Fax: +45 35398766; Email: garred{at}post5.tele.dk

Received June 22, 2007; Accepted August 25, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Polymorphisms in the MBL2 gene, which affect the structure and influence on the serum concentration of mannose-binding lectin (MBL), are associated with inflammatory and infectious conditions. The importance of MBL2 polymorphisms on outcome in critical ill patients is unclear. Five hundred and thirty-two consecutive critically ill patients admitted to an intensive care unit (ICU) were included over a period of 18 months. Five hundred and thirty-three individuals served as controls. Vital status was obtained 15.5 months after the last patient was included. MBL2 polymorphisms were determined by a PCR-based assay. Homozygosity for MBL2 variant alleles (O/O) causing MBL structural defects was associated with the highest adjusted mortality rate followed by homozygosity for the normal MBL2 allele (A/A) encoding high MBL levels, whereas heterozygous A/O patients had the most favourable outcome (P = 0.015). MBL2 alleles were not associated with death in ICU (n = 166, P = 0.7), but the association appeared soon after discharge from ICU (n = 366): hazard ratio (HR) for O/O using A/A as reference was 1.33 (95% CI: 0.8–2.2) and for A/O it was 0.62 (95% CI: 0.4–0.8) respectively (P = 0.0045) at completion. No difference in MBL2 frequency was observed between patients and controls at baseline, and between patients classified as having sepsis or not. However, patients with the MBL2 O/O genotype had an increased frequency of Gram-positive bacterial infection (P = 0.01). Heterozygosity for MBL2 alleles confers a protective effect whereas homozygosity is associated with the worst outcome soon after discharge from ICU. This may be an example of heterosis.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
Systemic inflammation and sepsis are the primary causes of death in intensive care units (ICU) despite advances in the treatment regimes over the recent years, latest with the guidelines of ‘The Surviving Sepsis Campaign’ from 2004 (1). The SOAP study from European ICUs showed that >35% of the patients were classified as having sepsis at some point during their ICU stay with a mortality rate of 27%, rising to >50% in patients with septic shock (2).

Even though the pathophysiology of systemic inflammation and sepsis is complex, it has long been recognized that inherited traits influence the individual ability to respond and resist appropriately to uncontrolled inflammation and infection (3). One of the genetically determined factors that have been suggested to be involved in systemic inflammation and sepsis is mannose-binding lectin (MBL) (4).

MBL is a liver-derived serum protein, which acts as a pattern-recognition molecule by binding to mannose and N-acetyl glucosamine containing molecular patterns present on various microorganisms (reviewed in 5). Moreover, MBL is also involved in sequestration of endogenous waste material and plays an important role in tissue homeostasis (6). In serum MBL is associated with the so-called MBL-associated serine proteases (MASPs) enabling activation of the complement system (7). Human MBL is derived from a single gene located on chromosome 10 (MBL2). Inter-individual differences in MBL serum concentration are mainly caused by structural variant alleles (B, C and D, at codons 54, 57 and 52, respectively) in the MBL2 gene which compromise assembly of MBL oligomers leading to a decrease in the absolute serum concentration as well as in the functional activity of the protein (reviewed in 8). Of particular interest is the fact that heterozygosity for MBL2 variant alleles causes in average a 85–90% drop in the serum concentration of functional MBL compared with the normal genotype while homozygous for the structural variant alleles are devoid of functional MBL (9). The normal MBL2 allele is named A, and the common designation for the variant alleles is O. In addition to the effect of the structural allelic variants, differences in MBL serum levels are determined by polymorphic sites in the promoter region of the MBL2 gene (10). In particular, a base substitution at codon-221 (G to C; promoter allele X) is associated with a lower MBL serum concentration. Presence of MBL2 polymorphisms is associated with the increased risk of respiratory infections during early childhood, especially during the first 6–18 months of life (11), and in patients with a concomitant immunodeficiency or severe disease (12–14). However, the MBL2 variant alleles are very frequent in different population around the world (8). Therefore, we and others have speculated whether the high frequency of these alleles are promoted by selective advantages of being heterozygous in analogy with the sickle cell haemoglobin S allele and malaria (9,15,16).

Mice devoid of MBL have been generated by knock out technology (17). However, in contrast to the human situation, mice have two functional MBL genes. In a sepsis model in which both MBL genes were deleted, it was shown that MBL offered protection against Staphyloccous aurus when bacteria were inoculated directly into the blood stream (17). By contrast in a more physiological model using the cecal ligation and puncture procedure to induce sepsis in which only one of the mice MBL genes were deleted paradoxically the gene targeted mice where more protected against death than the wild-type littermates (18). In another partial MBL knock out mice model, blood borne nematode Brugeria malayi microfilaria survived significantly longer than their wild-type counterparts (19). However, no differences in cytokine responses were observed. These results clearly indicate a dual and complex role of MBL in relation to systemic inflammation and infections. The importance of MBL as a susceptibility and modifying factor in humans for the development of sepsis and later mortality has been examined in the past years (20,21). These results indicate that the presence of MBL2 variant alleles determining low serum levels of MBL have been associated with the development of sepsis, and weakly associated to fatal outcome in adult patients admitted to intensive care and in children with the systemic inflammatory response syndrome (SIRS). In a recent British study, it was observed that MBL2 variant alleles are more common in adults with severe sepsis and septic shock than in normal population controls but no demonstrable influence on outcome was seen (22).

The conflicting results and the fact that MBL appears to play a complex role in SIRS and sepsis led us to investigate whether MBL2 alleles may be associated with outcome in prospectively enrolled patients admitted to intensive care.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
During the 18 months study period, 680 patients were admitted to the ICU and of these,109 were excluded because of exclusion criteria (Fig. 1). Of the resulting 571 ready for inclusion, a total of 547 were included in the study corresponding to an inclusion fraction of 96.0%. We were able to perform complete MBL2 genotyping in 532 patients accounting for 97.25% of the included patients.


Figure 1
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  		Figure 1. Flow diagram of study design and patient selection for case-control study.

 
More than 99% of the included patients were of Caucasian origin. At inclusion, genotype frequencies did not differ from those predicted by the Hardy–Weinberg expectations (P > 0.55). No significant differences in MBL2 structural and promoter genotype frequencies were observed between patients and 533 population controls (P > 0.8) (Table 1).


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  		Table 1. MBL2 genotypes, structural alleles, comparisons of patients without sepsis and those with sepsis, severe sepsis and septic shock

 
Of the 547 patients included in the study, 513 (93.8%) met the criteria for SIRS at the first date in the ICU and of these 75.4% met the criteria for sepsis. Of the 387 patients with sepsis, 199 (51.4%) met the criteria for severe sepsis and 130 (65.3%) of these met the criteria for septic shock. Of the 532 genotyped patients, 500 (94.0%) met the criteria for SIRS at the first date in the ICU and of these 75.2% met the criteria for sepsis (Fig. 1). Of the 376 patients with sepsis, 191 (50.8%) met the criteria for severe sepsis and 125 (65.4%) of these met the criteria for septic shock. No significant difference in MBL2 genotypes between the different SIRS-sepsis groups at admission was observed (P > 0.8) (Table 1). Because no significant difference was observed in MBL2 genotype frequencies between patients classified as having SIRS and Non-SIRS, we combined these groups in the subsequent analyzes.

Baseline characteristics and admission diagnosis are outlined in Table 2. There was no significant difference in age, type of admission, earlier disease or not, diagnosis and smoking habits at admission to the ICU stratified to the MBL2 genotypes. However, in the distribution of sex, patients classified as being immunosuppressed or not and active cancer (subgroup of earlier disease) were significantly different stratified according to the MBL2 genotypes. A logistic regression model using sepsis as a dependent parameter and taking into account the skewed parameters (gender, immunosuppression and active cancer), the MBL2 structural alleles were still not associated with the sepsis diagnosis (P > 0.47).


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  		Table 2. Baseline characteristics in the 532 included patients classified MBL2 structural variant alleles

 
Univariate analysis on all included patients during total follow-up until death or censoring showed that MBL2 structural genotype, admission type, previous disease, immunosuppression, sepsis at first date, age and first day sequential organ failure assessment (SOFA) score (Table 3) were significantly related to mortality. Gender, tobacco usage as pack years and weight were not independently related to mortality. The association with MBL2 genotypes was independent of cofactors that showed a significant association with fatal outcome in the univariate analysis when they were used in an expanded Cox-regression survival model including the parameters that were predictive in the univariate analysis (Table 3). SOFA score and sepsis at first date were not included in the same model because of the close correlation with each other. However, replacing SOFA score with sepsis at first date in this model revealed that the MBL2 results were independent of the sepsis diagnosis (data not shown).


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  		Table 3. Univariate analysis of categorical and continuous data for mortality risk in the total observation period

 
Kaplan–Meier survival curve analysis showed that O/O homozygotes were associated with the worst outcome, followed by those with the normal A/A genotype, whereas the heterozygous A/O genotype was associated with the best outcome (log rank, P = 0.0404) (Fig. 2A). It appeared from the survival curve that the MBL2 association was somewhat delayed compared to admission to ICU, thus we investigated at what time point the MBL2 association appeared. Censoring patients after discharge from ICU revealed that there was no association with the MBL2 variant alleles during stay at ICU (log rank, P = 0.73) (data not shown). However, the MBL2 association appeared immediately after discharge from ICU and became increasingly more apparent during follow-up when the baseline used was discharge from ICU (log rank, P = 0.0098) (Fig. 2B).


Figure 2
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  		Figure 2. (A) Kaplan–Meier survival plot of all 532 included patients during follow-up by the MBL2 genotype (log rank P = 0.0404). (B) Kaplan–Meier survival plot from departure from ICU in 366 patients alive at departure by the MBL2 genotype (log rank P = 0.0098).

 
In order to pin point in more detail at what time point the MBL2 association occurred, we performed the additional Cox regression analyses in a more restricted model after admittance to the ICU corrected for age, gender (included because of the MBL2/gender skewing in the demographic data in Table 1) and the first day SOFA score (Table 4) with censoring at different time points. It was revealed that the MBL2 alleles overall were independently and significantly associated with survival after 360 days observation period (P = 0.023) and after completion of the follow-up period (P = 0.015). Detailed analysis showed that using the A/A genotype as reference, the A/O genotype was associated with a significantly better outcome, whereas the O/O genotype was non-significantly associated with the worst outcome compared with the A/A genotype (Table 4). The hazard ratio (HR) for the A/O genotype compared with the A/A genotype was 0.72 (95% CI: 0.6–0.9) (P = 0.0059) for the total observation period, but the tendency was already seen from day 28. Essentially, the same results were seen if sepsis/no sepsis was used in the model instead of the first day SOFA score (data not shown).


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  		Table 4. Mortality risk and hazard ratio with 95% CI in all 532 patients with a determined MBL2 structural variant genotype (adjusted for MBL2, age, gender and SOFA score)

 
Based on the observation from the Kaplan–Meier plot, we stratified the patients whether they survived stay at intensive care or not. As expected, MBL2 alleles were not independently associated with outcome during stay at ICU when adjusted for the parameters used in Table 4 (data not shown).

The above results indicated influence of a delayed MBL2 dependency on mortality and we made a post hoc analysis in the 366 patients (68.8%) alive at departure from the ICU with a determined MBL2 genotype (Table 5). In this group, MBL2 genotypes were significantly associated with survival already at day 28 after admittance to the ICU with a HR for death for A/O of 0.64 (95% CI: 0.4–1.1) and 1.61 (95% CI: 0.8–3.2) for O/O (P = 0.0477) compared with the A/A genotype, while the corresponding figures at completion of follow-up were 0.62 (95% CI: 0.4–0.8) and 1.33 (95% CI: 0.8–2.2), respectively (P = 0.0045). Of the individual genotypes, only heterozygosity (A/O) deviated significantly from the A/A genotype, whereas O/O did not (Table 5). Essentially, the same results were observed when we used departure from ICU as baseline and discharge from hospital as censoring time (Table 5) and also total follow-up again with departure from ICU as baseline (P = 0.0045) (data not shown). Further analysis using the same Cox-regression model, but including the MBL2 promoter alleles with the highest expressing MBL2 genotype YAYA as reference showed that this model also was significant regarding MBL2 alleles (P = 0.0392) and that heterozygosity for the structural alleles were associated with a significant better outcome independently of the promoter alleles: XA/O, HR 0.44 (95% CI: 0.22–0.86), P = 0.0163 and YA/O, HR 0.661 (95% CI: 0.44–0.98), P = 0.0426. None of the other genotypes YA/XA, XA/XA and O/O, respectively, were significantly associated with either reduced or increased survival compared with the YA/YA reference genotype (P > 0.37).


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  		Table 5. Mortality risk and hazard ratio in 366 patients alive at departure from ICU with a determined MBL2 genotype (adjusted for MBL2 genotype, age, sex and 1 day SOFA score)—baseline time was admittance to the ICU excepta in which baseline was departure from ICU and censoring hospital discharge

 
Microbial specimens obtained at admission to the ICU were culture positive for 242 (44.2%) of the patients (Table 6). 98.2% of the included had blood specimen taken and of these were 15.1% culture positive. 68.0% of the included had tracheal or expectorate taken and of these were 40.3% culture positive. 87.9% of the patients had urine specimen taken and of these were 16% culture positive. At admission to the ICU 54.7% (n = 173) of the patients with the A/A genotype had one or more of the following infections; pneumonia, bacteraemia or wound infection, 54.6% with the A/O genotype and 51.6% with the O/O genotype. The results positive for coagulase negative Staphylococci were not displayed in the table with admissions cultures, since it is usually an insignificant result, if not re-cultured in a later culture from the same place. The MBL2 homozygous O/O defect genotype was associated primarily with Gram-positive bacteria since one or more of the O/O individual patient cultures were Gram-positive in 72.2% (8/11) of the positive cultures compared with 28% (21/75) in the A/O genotype and 37.4% (52/139) in the A/A genotype (P = 0.01), while no significant difference was observed for Gram-negative and anaerobe bacteria or fungi.


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  		Table 6. Microbial species (-coagulase-negative Staphylococci) diagnosed in cultures (blood, tracheal and urine) on admission to ICU in 532 MBL2 genotyped patients

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 FUNDING
 REFERENCES
 
This study shows that heterozygosity for the MBL2 structural genotypes (A/O) was significantly associated with improved survival compared with the normal A/A genotype and the homozygous variant O/O genotype after follow-up (censoring) period following admittance to the ICU. This association was independent of SOFA score, age and gender and the SIRS-sepsis consensus criteria on the first day in the ICU indicating that low and intermediate serum levels of MBL may confer a survival advantage in response both to severe infections and acute inflammation. Heterozygous advantage (heterosis) of MBL2 variant alleles was originally suggested 15 years ago when we and other researchers proposed that the high frequency of these alleles occurs due to a selective pressure promoting heterozygosity (9,15,16,23). Several studies have attempted to find molecular signatures whether the MBL2 gene could be subject to evolutionary selection with varying and inconsistent results (24–27). However, neither of these studies did test such a hypothesis in a disease setting, which might simulate an epidemic situation. Although far from ideal illness demanding ICU does at least to some extent provide such a situation and may give some conceptual clues. Nevertheless, at inclusion in the study, the MBL2 genotypes adhered closely to the Hardy–Weinberg expectations. However, during the observation period, an increasingly deviation towards an excess of A/O heterozygotes (present both in the YA/O and XA/O genotype situation) with a depletion of both homozygous A/A and O/O was observed supporting the notion that such events could take place in an epidemic situation. A mechanistic explanation to this observation could be that low and intermediate levels of functional MBL (A/O) would confer a relative advantage both in terms of optimal antimicrobial activity and less proinflammatory effect. On the other hand, deficiency of functional MBL (O/O) will make the patients more prone to become severely infected, while high levels of functional MBL (A/A) could directly be associated with the proinflammatory adverse effect following uncontrolled complement activation that particularly has been shown for down stream components such as C5a (28).

Nevertheless, from a clinical perspective, the main and most important finding that may be derived from this study was the observation that the MBL2 alleles almost immediately after discharge from the ICU and even during hospital stay appeared to be associated with either increased or decreased protection against death, whereas no such effect was seen during stay in the ICU. Our interpretation of this finding is that the combination of optimal surveillance and treatment combined with the fact that in a number of patients admitted to intensive care withdrawal of active life support to terminal patients is allowed might mask the MBL2 effect in relation to survival. Thus, we hypothesize that after intensive surveillance and treatment are halted and the patients are referred to their respective departments with less intensive treatment, the MBL2 effect may become apparent. The Kaplan–Meier curves show that particularly those homozygous for the variant alleles (O/O) might have the highest risk. In addition, because the MBL2 effect appears to be independent of SOFA score and sepsis diagnosis, which by themselves are important prognostic markers, it may be helpful to add MBL2 genotyping to the armament of parameters for prognostic staging of ICU patients. In a previous smaller study including 272 ICU patients (21), we observed that the MBL2 O/O genotype also had a negative impact on survival after ICU stay and that this was independent of the so-called simplified acute physiology score II (SAPSII) and sepsis diagnosis, but no A/O advantage could be detected. When MBL serum concentration was measured in a prospective insulin treatment study in patients that survived at least 5 days in ICU, it was observed that those functionally deficient of MBL, which did not receive insulin had an increased mortality rate consistent with the present findings (29). In other studies addressing MBL and ICU survival only mortality related to ICU stay have been reported (22,30). In these studies, MBL2 polymorphisms were related to sepsis and/or infections but not related to increased mortality during ICU stay in agreement with the present findings. However, in a recent American study, patients homozygous for the B variant had greater likelihood of septic shock and development of acute respiratory distress syndrome (ARDS) (31). In the subgroup of patients with ARDS, an increased risk of death was observed after 60 days for B/B homozygotes.

We found that the frequency of the MBL2 alleles at inclusion was similar as the one in Danish population controls consistent with our previous study (21) and the frequency of MBL2 variant alleles was not significantly different between ICU patients classified having sepsis or not or which developed severe sepsis and septic shock which is in variance with our former study. Nearly, the same prevalence of positive bacterial cultures at admission to the ICU was observed between the different MBL2 genotypes. However, further analysis revealed that the MBL2 O/O genotype indeed was associated with the increased incidence of Gram-positive bacteria. The possible association between Gram-positive bacteria and the MBL2 O/O genotype has also been observed in other studies (32). Nevertheless, MBL2 variant alleles have also repeatedly been shown to be associated with Gram-negative infections (21,33). The reason why some studies find an association between MBL deficiency and different bacterial isolates probably derives from the fact that many clinically isolates do not bind MBL and differences in the percentage of positive isolates that may be obtained. Another reason for the diversity between the different studies may rely on the fact that MBL also function as a scavenger molecule in maintenance of internal tissue homeostasis and that apparent MBL associations may be due to disturbances in this scavenger system rather than a direct anti-infectious association (6). Moreover, the different patient populations are probably very heterogeneous even though we try to use objective SIRS/sepsis criteria in order to compare.

It would be tempting to supplement ICU patients with purified or recombinant MBL, which have been used in pilot studies both to patients with infections and healthy volunteers with no adverse effects (34,35). Recombinant MBL are now in phase 2 trials in order to explore the putative anti-infectious effect of MBL in patients with chemotherapy-related neutropenia (www.enzon.com). However, the results from the present study cast doubts of the use of MBL in ICU patients with systemic inflammation and sepsis, but if used only through a narrow therapeutical window.

Although the general relevance of cohorts studies can always be discussed, the present study provide a number of advantages. First, it is a prospective and almost complete study in, which more than 97% of the patients fulfilling the inclusion criteria over at 18 months period were genotyped, which minimize the risk for selection bias. Secondly, it is larger compared with the earlier published MBL studies in ICU populations, which reduces the chance for a type I error and thirdly the population is an ethnically homogenous Caucasian population. The primary limitation is the 15 included patients with an unknown MBL2 genotype (2.7%), but this is a small number and the mortality for this group was not different from the other 532 with a determined MBL2 genotype. We chose to use the MBL2 genotypes instead of measuring the concentration of MBL serum on day one because of the very good correlation between the MBL2 genotypes and the concentration and function of MBL and our previous experience showing the strength using MBL2 genotypes in disease association studies (36).

In conclusion, the present study has shown that ICU patients heterozygous for MBL2 variant alleles are partly protected from fatal outcome after ICU stay, but more importantly that this effect becomes apparent shortly after discharge from ICU and may thus go unnoticed by the caring physicians.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
FUNDING
 REFERENCES
 
Setting
Herlev Hospital in Copenhagen, Denmark is a university hospital with 568 beds. The central mixed surgical–medical ICU serves the medical and surgical blocks of the hospital. The ICU is a six-bed unit with exclusively single-bed rooms.

Study population and design
A schematic summary of the study design, inclusion and exclusion criteria is presented in Figure 1. All adult patients (≥18 years) admitted to the ICU between December 2, 2002 and June 1, 2004 (18 months) were screened for inclusion. Patients readmitted who had been included on their first admission were only registered according to the first admission. Other exclusion criteria were admissions for the sole purpose of fenytoin loading and broncho-alveolar lavage (BAL), constraint commitment because of psychiatric disease, fault referral and exclusive stay in the post anaesthesia care unit due to overbooking of the ICU. The Institutional Review Boards of the County of Copenhagen approved the protocol (file number KA 02071). Informed consent was obtained from the patients or from their relatives.

All data were initially recorded in case-record forms (CRF) by the ICU physicians on rounds, physicians from the project group or by the coordinating investigator. At admission to ICU baseline clinical information concerning underlying disease, cause of admission, tobacco consumption, immunosuppressive factors, infection on admission and admission diagnosis were recorded in the CRF. Chest radiography was usually performed at admittance. Bacterial cultures were taken on admission from blood, nasopharynx, urine and tracheal aspirate or expectorate. Cultures were processed according to standardized methods on the Department of Clinical Microbiology. Organ failure at admission was defined according to the ‘worst value’ of the first day SOFA score (37). The SIRS-Sepsis criteria were recorded (38) in the five classes; none, SIRS, sepsis, severe sepsis and septic shock. From the Central Office of Civil Registration in Denmark, we requested and received a vital status for all included patients at the 14th of September 2005; if death had occurred the date of death was registered.

Controls
As control subjects served 533 randomly selected individuals (mean age 50 years, range 18–67) from the Danish population by means of the Civil Registration System (39).

Previous disease (underlying disease)
One or more of the following conditions were recorded: acute myocardial infarction (AMI), congestive heart disease, diabetes mellitus types I and II, Acute pancreatitis, chronic pancreatitis, hepatic disease, renal disease, chronic obstructive pulmonary disease (COPD), active malignant disease, recent trauma.

Immunosuppressive factors
One or more of the following conditions: diabetes mellitus, cirrhosis of the liver, immunosuppressive agents such as the use of high doses of corticosteroids, alcohol or drug abuse, active malignant disease, or renal failure (Se-creatinine ≥200 mol/l) were registered.

Tobacco consumption
Patients divided in the current smoking, ex-smokers, possible ex-smokers, never smoked, were not known. Number of pack years was registered. One pack year is equal to smoking of 20 cigarettes daily in 1 year.

Summary of classification criteria (SIRS, sepsis, severe sepsis and septic shock)
SIRS, sepsis, severe sepsis and septic shock and none were defined and registered in accordance with the recommendations of the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference (38).

Genotyping methods
Blood collected in 10 ml EDTA containing vacutainers was collected for DNA extraction. Anticoagulated blood was frozen at –80°C before processing. MBL2 single nucleotide polymorphisms (SNPs) in the form of the structural variants named B (codon 54, rs1800450, C (codon 57, rs1800451) and D (codon 52, rs5030737) as well as the regulatory variants named H/L (–550, rs11003125), X/Y (–221, rs7096206) and P/Q (+4, rs7095891) were typed by PCR using sequence specific priming (PCR-SSP) as previously described (21). Although, the typing was performed as SNP-typing, the results were combined in haplotypes, based on strong linkage disequilibrium between the SNPs that gives the seven known major MBL2 haplotypes: four functional haplotypes; LXPA, LYPA, LYQA, and HYPA (the normal allele is designated ‘A’) and three defective haplotypes; LYPB, LYQC, and HYPD. All the three structural variant alleles (B, C and D) have a considerable effect on MBL concentrations and to avoid small groups, the three alleles were grouped in one category called allele 'O' for statistical analyses. Likewise, for statistical analyses, we only included the X/Y promoter variation at position –221. The X variant is always found on a functional haplotype (LXPA) and has been shown to have a down regulating effect on MBL expression. Thus, the following six MBL2 genotypes/haplotypes were defined: the A/A group, two normal structural alleles with high-expression promoter activity in position –221 (YA/YA) or one high-expression promoter and one low-expression promoter (YA/XA) or two low-expression promoters (XA/XA); the A/O group, one variant structural allele (i.e. defective allele) and one normal structural allele regulated by a high-expression promoter (YA/O) or a low-expression promoter (XA/O) and the O/O group with two defective structural alleles.

Statistical analyses
Contingency table analyses and Fisher’s exact test were used to compare frequencies. Deviation from the Hardy–Weinberg expectations was tested by simple gene counting using the {chi}2 test for comparing observed and expected values. Kruskall–Wallis or Mann–Whitney tests were used to compare continuous data. Log-rank test and Kaplan–Meier curves were used to estimate survival. When appropriate, logistic regression and Cox regression multivariate survival analyses were performed. Only two-sided tests were used.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
REFERENCES
 
D.H. was supported by a PhD grant from the County of Copenhagen and by grants from Consultant Johan Boserup and Lise Boserups Foundation; The Foundation of the Danish Society of Anesthesiology and Intensive Care; Professor Sophus H. Johansens Foundation of the 23rd of august 1981, The Foundation of Director Jacob Madsen and wife Olga Madsen, The Novo Nordisk Research Foundation, The Benzon Foundation, The Danish Medical Research Council and Rigshospitalet.


   ACKNOWLEDGEMENTS
 
We gratefully acknowledge the assistance from the staff at the ICU at Herlev Hospital and especially we wish to express our gratitude for the excellent technical assistance from Mss Ulla Gregersen, Bente Lauersen and Birgit Edelfors at Herlev Hospital and Bente Frederiksen and Vibeke Weirup at Rigshospitalet and secretarial assistance from Hannah Brostrøm who in a large part keyed the data material. Moreover, we wish to thank Peter Bjørn Jensen who organized the databases and Dr Per Winkel for statistical advice both at Rigshospitalet. Finally, we wish to express our gratitude to the Department of Clinical Microbiology for processing the cultures and the Department of Clinical Biochemistry, both at Herlev Hospital for handling blood samples in weekends. Conflict of Interest statement. None declared.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 FUNDING
 REFERENCES
 

  1. Dellinger R.P., Carlet J.M., Masur H., Gerlach H., Calandra T., Cohen J., Gea-Banacloche J., Keh D., Marshall J.C., Parker M.M., et al. Surviving Sepsis Campaign guidelines for management of severe sepsis and septic shock. Crit. Care Med. (2004) 32:858–873.[CrossRef][Web of Science][Medline]

  2. Vincent J.-L., Sakr Y., Sprung C.L.M., Ranieri V.M., Reinhart K., Gerlach H., Moreno R., Carlet J., Le Gall J.-R., Payen D., et al. Sepsis in European intensive care units: results of the SOAP study. Crit. Care Med. (2006) 34:344–353.[CrossRef][Web of Science][Medline]

  3. Sørensen T.I.A., Nielsen G.G., Andersen P.K., Teasdale T.W. Genetic and environmental influences on premature death in adult adoptes. N. Engl. J. Med. (1988) 318:727–732.[Abstract]

  4. Garred P., Madsen H.O. Genetic susceptibility to sepsis: a possible role for mannose-binding lectin. Curr. Infect. Dis. Rep. (2004) 6:367–373.[CrossRef][Medline]

  5. Dommett R.M., Klein N., Turner M.W. Mannose-binding lectin in innate immunity: past, present and future. Tissue Antigens (2006) 68:193–209.[CrossRef][Web of Science][Medline]

  6. Nauta A.J., Raaschou-Jensen N., Roos A., Daha M.R., Madsen H.O., Borrias-Essers M.C., Ryder L.P., Koch C., Garred P. Mannose-binding lectin engagement with late apoptotic and necrotic cells. Eur. J. Immunol. (2003) 33:2853–2863.[CrossRef][Web of Science][Medline]

  7. Thiel S., Vorup-Jensen T., Stover C.M., Schwaeble W., Laursen S.B., Poulsen K., Willis A.C., Eggleton P., Hansen S., Holmskov U., et al. A second serine protease associated with mannan-binding lectin that activates complement. Nature (1997) 386:506–510.[CrossRef][Medline]

  8. Garred P., Larsen F., Seyfarth J., Fujita R., Madsen H.O. Mannose-binding lectin and its genetic variants. Genes Immun. (2006) 7:85–94.[CrossRef][Web of Science][Medline]

  9. Garred P., Thiel S., Madsen H.O., Ryder L.P., Jensenius J.C., Svejgaard A. Gene frequency and partial protein characterization of an allelic variant of mannan binding protein associated with low serum concentrations. Clin. Exp. Immunol. (1992) 90:517–521.[Web of Science][Medline]

  10. Madsen H.O., Garred P., Thiel S., Kurtzhals J.A., Lamm L.U., Ryder L.P., Svejgaard A. Interplay between promoter and structural gene variants control basal serum level of mannan-binding protein. J. Immunol. (1995) 155:3013–3020.[Abstract]

  11. Koch A., Melbye M., Sørensen P., Homøe P., Madsen H.O., Mølbak K., Hansen C.H., Andersen L.H., Hahn G.W., Garred P. Acute respiratory tract infections and mannose-binding lectin insuffiency during early childhood. JAMA. (2001) 285:1316–1321.[Abstract/Free Full Text]

  12. Garred P., Madsen H.O., Hofmann B., Svejgaard A. Increased frequency of homozygosity of abnormal mannan-binding-protein alleles in patients with suspected immunodeficiency. Lancet (1995) 346:941–943.[CrossRef][Web of Science][Medline]

  13. Garred P., Pressler T., Madsen H.O., Frederiksen B., Svejgaard A., Høiby N., Schwartz M., Koch C. Association of mannose-binding lectin gene heterogeneity with severity of lung disease and survival in cystic fibrosis. J. Clin. Invest. (1999) 104:431–437.[Web of Science][Medline]

  14. Neth O., Hann I., Turner M.W., Klein N.J. Deficiency of mannose-binding lectin and burden of infection in children with malignancy: a prospective study. Lancet (2001) 358:614–618.[CrossRef][Web of Science][Medline]

  15. Garred P., Harboe M., Oettinger T., Koch C., Svejgaard A. Dual role of mannan-binding protein in infections: another case of heterosis? Eur. J. Immunogenet. (1994) 21:125–131.[Web of Science][Medline]

  16. Lipscombe R.J., Sumiya M., Hill A.V.S., Lau Y.L., Levinsky R.J., Summerfield J.A., Turner M.W. High frequencies in African and non-African populations of independent mutations in the mannose binding protein gene. Hum. Mol. Genet. (1992) 1:709–715.[Abstract/Free Full Text]

  17. Shi L., Takahashi K., Dundee J., Shahroor-Karni S., Thiel S., Jensenius J.C., Gad F., Hamblin M.R., Sastry K.N., Ezekowitz R.A. Mannose-binding lectin-deficient mice are susceptible to infection with Staphylococcus aureus. J. Exp. Med. (2004) 199:1379–1390.[Abstract/Free Full Text]

  18. Takahashi K., Gordon J., Liu H., Sastry K.N., Epstein J.E., Motwani M., Laursen I., Thiel S., Jensenius J.C., Carroll M., et al. Lack of mannose-binding lectin-A enhances survival in a mouse model of acute septic peritonitis. Microb. Infect. (2002) 4:773–784.[CrossRef][Web of Science][Medline]

  19. Carter T., Sumiya M., Reilly K., Ahmed R., Sobieszczuk P., Summerfield J.A., Lawrence R.A. Mannose-binding lectin A-deficient mice have abrogated antigen-specific IgM responses and increased susceptibility to a nematode infection. J. Immunol. (2007) 178:5116–5123.[Abstract/Free Full Text]

  20. Fidler K.J., Wilson P., Davies J.C., Turner M.W., Peters M.J., Klein N.J. Increased incidence and severity of the systemic inflammatory response syndrome in patients deficient in mannose-binding lectin. Int. Care Med. (2004) 30:1438–1445.[Web of Science][Medline]

  21. Garred P., Strom J.J., Quist L., Taaning E., Madsen H.O. Association of mannose-binding lectin polymorphisms with sepsis and fatal outcome, in patients with systemic inflammatory response syndrome. J. Infect. Dis. (2003) 188:1394–1404.[CrossRef][Web of Science][Medline]

  22. Gordon A.C., Waheed U., Hansen T.K., Hitman G.A., Garrard C.S., Turner M.W., Klein N.J., Brett S.J., Hinds C.J. Mannose-binding lectin polymorphisms in severe sepsis: relationship to levels, incidence, and outcome. Shock (2006) 25:88–93.[Web of Science][Medline]

  23. Garred P., Madsen H.O., Kurtzhals J.A.L., Lamm L.U., Thiel S., Hey A.S., Schade F.U., Svejgaard A. Diallelic polymorphism may explain variations of blood concentration of mannan-binding protein in Eskimos, but not in black Africans. Eur. J. Immunogen. (1992) 19:403–412.[Web of Science][Medline]

  24. Bernig T., Taylor J.G., Foster C.B., Staats B., Yeager M., Chanock S.J. Sequence analysis of the mannose-binding lectin (MBL2) gene reveals a high degree of heterozygosity with evidence of selection. Genes Immun. (2004) 5:461–476.[CrossRef][Web of Science][Medline]

  25. Seyfarth J., Garred P., Madsen H.O. The ‘involution’ of mannose-binding lectin. Hum. Mol. Genet. (2005) 14:2859–2869.[Abstract/Free Full Text]

  26. Verdu P., Barreiro L.B., Patin E., Gessain A., Cassar O., Kidd J.R., Kidd K.K., Behar D.M., Froment A., Heyer E., et al. Evolutionary insights into the high worldwide prevalence of MBL2 deficiency alleles. Hum. Mol. Genet. (2006) 15:2650–2658.[Abstract/Free Full Text]

  27. Walsh E., Sabeti P., Hutcheson H., Fry B., Schaffner S., de Bakker P., Varilly P., Palma A., Roy J., Cooper R., et al. Searching for signals of evolutionary selection in 168 genes related to immune function. Hum. Genet. (2006) 119:92–102.[CrossRef][Web of Science][Medline]

  28. Ward P.A. The dark side of C5a in sepsis. Nat. Rev. Immunol. (2004) 4:133–142.[CrossRef][Web of Science][Medline]

  29. Hansen T.K., Thiel S., Wouters P.J., Christiansen J.S., Van den Berghe G. Intensive insulin therapy exerts antiinflammatory effects in critically ill patients and counteracts the adverse effect of low mannose-binding lectin levels. J. Clin. Endocrinol. Met. (2003) 88:1082–1088.[Abstract/Free Full Text]

  30. Sutherland A.M., Walley K.R., Russell J.A. Polymorphisms in CD14, mannose-binding lectin, and Toll-like receptor-2 are associated with increased prevalence of infection in critically ill adults. Crit. Care Med. (2005) 33:638–644.[CrossRef][Web of Science][Medline]

  31. Gong M.N., Zhou W., Williams P.L., Thompson B.T., Pothier L., Christiani D.C. Polymorphisms in the mannose binding lectin-2 gene and acute respiratory distress syndrome. Crit. Care Med. (2007) 35:48–56.[CrossRef][Web of Science][Medline]

  32. Roy S., Knox K., Segal S., Griffiths D., Moore C.E., Welsh K.I., Smarason A., Day N.P., McPheat W.L., Crook D.W., et al. MBL genotype and risk of invasive pneumococcal disease: a case-control study. Lancet (2002) 359:1569–1573.[CrossRef][Web of Science][Medline]

  33. Smithson A., Munoz A., Suarez B., Soto S.M., Perello R., Soriano A., Martinez J.A., Vil J., Horcajada J.P., Mensa J., et al. Association between mannose-binding lectin deficiency and septic shock following acute pyelonephritis due to Escherichia coli. Clin. Vaccine Immunol. (2007) 14:256–261.[Abstract/Free Full Text]

  34. Garred P., Pressler T., Lanng S., Madsen H.O., Moser C., Laursen I., Balstrup F., Koch C., Koch C. Mannose-binding lectin (MBL) therapy in an MBL-deficient patient with severe cystic fibrosis lung disease. Pediatric. Pulmonol. (2002) 33:201–207.[CrossRef][Web of Science][Medline]

  35. Petersen K., Matthiesen F., Agger T., Kongerslev L., Thiel S., Cornelissen K., Axelsen M. Phase I safety, tolerability, and pharmacokinetic study of recombinant human mannan-binding lectin. J. Clin. Immunol. (2006) 26:465–475.[CrossRef][Web of Science][Medline]

  36. Garred P., Larsen F., Madsen H.O., Koch C. Mannose-binding lectin deficiency—revisited. Mol. Immunol. (2003) 40:73–84.[CrossRef][Web of Science][Medline]

  37. Ferreira F.L., Bota D.P., Bross A., Mélot C., Vincent L. Serial evaluation of the SOFA score to predict outcome in critically ill patients. JAMA (2001) 286:1754–1758.[Abstract/Free Full Text]

  38. Members of the American College of Chest Physicians/Society of Critical Care Medicine. American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference: Definitions for sepsis and organ failure and guidelines for the use of innovative therapies in sepsis. Crit. Care Med. (1992) 20:864–874.[Web of Science][Medline]

  39. Pedersen M., Jacobsen S., Garred P., Madsen H.O., Klarlund M., Svejgaard A., Pedersen B.V., Wohlfahrt J., Frisch M. Strong combined gene-environment effects in anti-cyclic citrullinated peptide-positive rheumatoid arthritis: a nationwide case-control study in Denmark. Arthritis. Rheum. (2007) 56:1446–1453.[CrossRef][Web of Science][Medline]


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