Mannose-binding lectin gene variants and infections in patients receiving autologous stem cell transplantation

Background Serious infections are common in patients undergoing autologous stem cell transplantation (ASCT) mainly because of the effects of immunosuppression. The innate immune system plays an important role in the defense against different infections. Mannose binding lectin (MBL) is a central molecule of the innate immune system. There are several promoter polymorphisms and structural variants of the MBL2 gene that encodes for this protein. These variants produce low levels of MBL and have been associated with an increased risk for infections. Methods Prospective cohort study. The incidence, severity of infections and mortality in 72 consecutive patients with hematologic diseases who underwent ASCT between February 2006 and June 2008 in a tertiary referral center were analyzed according to their MBL2 genotype. INNO-LiPA MBL2 was used for MBL2 gene amplification and genotyping. Relative risks (RR) (IC95%) as measure of association were calculated. Multivariate analysis was performed using logistic regression. Results A statistically significant higher number of fungal infections was found in patients with MBL2 variants causing low MBL levels (21.1%versus1.9%, p=0.016). In this MBL2 variant group infection was more frequently the cause of mortality than in the MBL2 wild-type group (p=0.05). Although not statistically significant, there was a higher incidence of major infections in the MBL2 variant group as well as a higher number of infections caused by gram-positive bacteria. Conclusions Low-producer MBL2 genotypes were associated with an increased number of fungal infections in ASCT patients, which would suggest that MBL has a protective role against such infections. ASCT patients with MBL2 variant genotypes are more likely to die as a result of an infection.


Background
Patients undergoing autologous stem cell transplantation (ASCT) are at a high risk of serious infection, mainly due to both the procedure and the underlying disease requiring the transplant, which in most patients undergoing the procedure is hematological [1][2][3][4][5].
Mannose-binding lectin (MBL) is a circulating liversynthesized C-type serum protein of importance in the innate immune defense system. It is one of the recognition molecules in the lectin complement activation pathway [6]. Human MBL is derived from a single gene located on chromosome 10 (MBL2) [3][4][5][6][7][8].
Serum levels and the function of MBL oligomers are strongly influenced by genetic variants [9]. Low levels of MBL have been associated with increased susceptibility to infections, especially in recurrent or severe forms, even in immunocompetent individuals [10]. However, isolated low MBL serum levels do not usually cause disease. This deficiency appears to be associated with severe infections when other immune deficiencies occur [11].
The effect of MBL deficiency in the infectious complications of patients receiving chemotherapy is variable [11]. It has been associated with serious infections and more prolonged episodes of febrile neutropenia [11][12][13], as well as with an earlier first severe infection in deficient patients [14]. There are also several studies which analyze the relationship between MBL and allogeneic stem cell transplantation infections [15][16][17][18][19][20][21][22], but until now, only two studies dealing with this issue in ASCT have been published [23,24]. The focus of this study is the association between MBL2 gene variants and the risk of infections in patients with different hematologic diseases undergoing ASCT.

Patients and methods Patients
A prospective cohort study of all patients who received an ASCT for hematologic disease from February 2006 to June 2008 in the Hospital Universitario Marqués de Valdecilla (1,000-bed teaching hospital) was performed. All transplanted patients >13 years old without immunodeficiency (solid tumors, HIV infection or congenital immunodeficiencies) were included. Patients were followed up after inclusion in the study for 6 months.

Infections
Intercurrent infections were monitored during the early period (0-30 days after ASCT), the intermediate period (30-100 days after ASCT) and the late period (>100 days after ASCT). Infections were prospectively collected by the clinical research associates from the institution using a standardized data collection form, following the protocols of the Hematology Department of the Hospital Universitario Marqués de Valdecilla, in compliance with the clinical guidelines of the Infectious Diseases Society of America [25]. Prophylactic measures included isolation in high-pressure rooms with air filters and the administration of intravenous acyclovir at prophylactic doses from day −3. Antibiotics and antifungal prophylaxis were not routinely administered.
An infectious episode was defined as a microbiologically or clinically documented infection. Microbiologically documented infections with the same organisms during the same phase of therapy occurring more than 7 days apart were counted as different infections. Major infection was predefined as either sepsis confirmed on microbiological testing or a systemic inflammatory response syndrome with highly suggestive radiographic or clinical characteristics and the requirement for a specific antimicrobial treatment in both cases. The following conditions were excluded: single positive blood culture resulting from contaminant skin bacteria, upper respiratory tract infections, culture-negative interstitial pneumonitis, dermatological varicella zoster reactivation, and local herpes simplex reactivation. Severe herpes virus infection was predefined as invasive viral infections requiring prompt treatment and hospitalization. A positive cytomegalovirus (CMV) blood culture or CMV-pp65 antigenemia assay or polymerase chain reaction (PCR) for CMV DNA (>400 copies/ml) (COBAS Amplicor CMV Monitor, Roche Diagnostics) and symptoms of organ dysfunction were required to fulfill the criteria of a clinically significant CMV infection/reactivation [26]. Episodes of asymptomatic CMV reactivation, primary varicella zoster virus and Epstein-Barr virus infections were excluded.

MBL2 genotyping
Blood was collected in EDTA-stabilized tubes in compliance with approved protocols from our institution. Genomic DNA from patients and controls was extracted from peripheral blood by using the Maxwell 16 Genomic DNA Purification system. For MBL2 gene amplification and genotyping, the INNO-LiPA MBL2 (Innogenetics Diagnostica Iberia S.L.U, Barcelona, Spain) was used, following the manufacturer's instructions. The INNO-LiPA MBL2 is a line probe assay, designed for genotyping the 6 variations in the human MBL2 gene (−550G > C, −221G > C, +4C > T, R52C, G54D, and G57E), providing an analysis of the seven common haplotypes and the 28 possible resulting diplotypes.
Exon mutations called structural variants (normal allele or "wild-type" A and mutant alleles B, C and D which together were called allele O) and promoter polymorphisms (H/L, Y/X and P/Q) were analyzed. Variants YA/YA, XA/ XA and YA/XA were classified as A/A. Those with any mutation in the exon such as YA/O, XA/O were classified as A/O and, lastly, individuals homozygous for the structural variants in the exon were considered as O/O. Thus, patients were divided in two groups according to their MBL2 genotype: 1) normal homozygous patients (AA) and 2) patients with AO and OO genotypes, who were considered to be lower MBL producers [24].

Statistical analysis
Results were analyzed according to MBL2 genotype, comparing normal homozygous patients (AA) and patients with AO and OO genotype and to determine if there were differences between the two groups in terms of demographic characteristics, underlying disease, infection parameters and mortality. Statistical analysis was performed using a two-tailed χ 2 test and a Fisher's exact test, Student t-test or Mann Whitney, as appropriate in each case. For m × n tables, a Fisher's exact test was estimated using the Monte Carlo method. Relative risks (RR) and their 95% confidence intervals (CI) were calculated to analyze the association between genetic variants and polymorphisms and the risk of concomitant infections as dependent variables (overall and by microorganisms). Multivariate analysis was performed using logistic regression to estimate the association between genetic variants and polymorphisms and the risk of underlying infections adjusted by confusion factors. A regression model was constructed for each type of infection, with infection-related variables being the outcome and the independent variable being the MBL2 genotypes, adjusted for sex, age, hematologic disease, active pre-transplant hematologic disease. A two-tailed P <0.05 was considered statistically significant. Data were analyzed using Stata (SE 10.0, Stata Corporation, College Station, TX) and SPSS (version 19) (SPSS Inc., Chicago, IL) statistical software.

Ethics
This study was approved by the Ethics Committee of the Autonomous Community of Cantabria, Spain. All patients (>18 years) signed informed consent to participate in the study. For children under 18 years, the parents had to sign informed consent.

MBL2 structural variants, promoter polymorphisms and genotypes
Seventy-two patients were included. Median age at the time of transplantation was 54 years . During the study period, no patients under 19 years underwent AST. Forty-two (42) patients were men. Patients were divided in two groups according to their MBL2 genotype: patients homozygous (AA) for wild type MBL2 (n = 53) and other patients, AO/OO (n = 19), which included heterozygous patients (AO) (n = 16), and homozygous patients (OO) (n = 3). No statistically significant differences were found in both groups for gender, age, distribution according to different types of hematologic disease or hematologic disease activity at the time of transplantation. Patient characteristics, underlying disease and number of previous lines of treatment are shown in Tables 1 and 2.  Table 3 shows infection characteristics. Sepsis was common (43.1%) and equally distributed between both groups, as was urinary tract infection which occurred at a lower frequency (11.1%) than expected. There was also a low incidence of pneumonia (5.6% of all patients), which occurred more frequently in the MBL2 variant group, 10   Regarding the type of infection, gram-positive bacteria were more frequent in the MBL2 variant group, 47.4% versus 26.4%, RR = 1.8 (95% CI: 0.9-3.4, p = 0.09). There was a greater incidence of infections caused by Staphylococcus The incidence of fungal infections in the variant MBL2 group (3 Candida albicans, 1 Mucor, 1 Aspergillus) was statistically significantly higher than in the MBL2 wild-type group (1 Candida spp), RR = 11.5 (95% CI: 1.3-93.7, p = 0.016), as confirmed in a multivariate analysis (Table 4).
There were no statistically significant differences in the incidence of infection caused by gram-negative bacteria, RR: 0.9 (95% CI: 0.4-1.9, p = 0.74), or by virus between both groups, RR: 2.8 (95% CI: 0.2-42.4, p = 0.46), although there was a higher incidence of viral infections in the variant MBL2 group.
More patients died in the variant MBL2 group than in the wild-type MBL2 group (15.8% versus 7.5% respectively, p = 0.37), although this difference was not statistically significant. However, all deaths in the variant MBL2 group were related to infection, whereas in the wild-type MBL2 group, deaths were due to other causes, with a p-value of 0.05 at the limit of statistical significance (Table 3).

Discussion
This prospective study focused on comparing the incidence of infection and mortality after ASCT between patients with the wild-type MBL2 genotype compared to patients carrying a variant MBL2 genotype. The MBL2 variant is associated with lower levels of serum MBL, so it may be related with a greater number of infections and/or more serious infections. A multivariate analysis was performed to rule out other factors that could cause a higher incidence or severity of infection (disease status-response vs. type of hematologic disease, active pre-transplant hematologic disease, sex and age).
The study group was homogeneous, with no differences in age, gender or hematologic disease, and presented similar disease activity at the time of transplantation.
So far, there are few studies describing the incidence of infections in ASCT related to MBL2 gene variants. Horiuchi et al. [23] in a series of 113 patients treated with high-dose chemotherapy and ASCT, most of whom had hematologic diseases, carried out a study of the incidence of major bacterial infection in patients with lowproducing genotypes compared to the wild-type genotype, finding an association between the low-producing genotypes and a highly increased and significant risk of major bacterial infections. The incidence of major bacterial infections (9%) was lower than that observed in the current study (18%). This could be related with the use of prophylactic antimicrobials including ciprofloxacin and fluconazole in all their patients, in contrast to the usual practice in our center.
Mølle et al. [24], in a retrospective study compared MBL2 gene variants with the risk of severe infections in multiple myeloma in patients receiving ASCT after highdose melphalan. They found that patients homozygous for wild-type MBL2 had a significantly reduced risk of septicemia compared to patients carrying the variant MBL2. In our series, no differences were found in the number of patients with sepsis between the two groups. Although the lack of association between these infections and MBL2 gene variants could be explained by the sample size, this could also be due to the different underlying diseases: in Mølle's study, all patients were diagnosed with multiple myeloma while our patients had heterogeneous hematologic diseases and therefore could have had a higher risk of infection. They also found more severe infections in patients with variant MBL2, although the differences were not statistically significant. Similarly, in our series more severe infections and a greater number of episodes of pneumonia were found in patients with variant MBL2.
When the incidence of infections caused by different microorganisms was analyzed, a higher number of infections caused by gram-positive bacteria was found in patients carrying the variant MBL2 form, and these were mainly staphylococci. In this respect, the incidence of infections  [27] in critically ill patients admitted to an intensive care, showed an association between the MBL2 O/O genotype with an increased incidence of gram-positive infections. However, in two different studies by Neth et al. [28] and Shang et al. [29], S. epidermidis generally demonstrated low MBL binding activity, which would contradict the hypothesis that MBL plays an important role in infections caused by S. epidermidis. One explanation that has been proposed for the diversity between the different studies may rely on the fact that MBL also functions as a scavenger molecule in maintaining internal tissue homeostasis. Apparent MBL associations may be due to disturbances in this scavenger system, rather than a direct antiinfectious effect [30].
In the current study, no differences were found in the incidence of infections caused by gram-negative bacteria in patients with wild-type MBL2 genotype versus patients carrying a variant MBL2 genotype. This is consistent with the studies by Hellemann [27] and Sutherland [31]. There was only one case of viral infection in each group; in the MBL2 variant group this was caused by CMV, a potentially very serious disease, while the virus in the MBL2 wild-type group was a reactivation of varicella zoster, which is more common in hematological patients.
Granell et al. [18] showed that MBL pathway dysfunction due to genetically determined deficiencies of MBL influence the outcome of allogenic stem cell transplantation by increasing susceptibility to fungal invasive infections. In patients with ASCT, Horiuchi et al. [23] found that 3 of the 16 patients with major infections who had a pulmonary aspergillosis were homozygous for A, i.e. the normal MBL allele. On the contrary, in our study, 4 patients with fungal infection carried the MBL2 deficient genotype; 2 had Aspergillus and Mucor pneumonia (both died as a consequence of their fungal infection); 1 had candidemia; and 1 had disseminated mucocutaneous Candida infection. Several studies have been published showing that MBL may play a crucial role in the innate immunity against yeast infections by increasing PMN uptake [32,33].
In our series, the incidence of mortality due to infection was higher in the MBL2 variant form, and the cause of death in all patients was major infection. These findings are in line with other groups of immunosuppressed patients who had low-producing MBL2 genotypes [27,34].

Conclusions
In summary, the results of our study suggest that ASCT patients with the MBL2 variant gene have increased fungal infections, which would suggest that MBL offers protection against such infections. It would be reasonable to assess whether early antifungal prophylaxis in our ASCT patients with the MBL2 variant gene could reduce the incidence of such infections. Due to the contradictory results of the previous reports, further studies are needed to clarify whether low-producing MBL2 genotypes could play a role in gram-positive infections, especially in S. epidermidis infections. ASCT patients with MBL2 variant genotypes are more likely to die as a result of an infection.