Mannose-binding lectin 2 polymorphisms do not influence frequency or type of infection in adults with chemotherapy induced neutropaenia.

BACKGROUND: Mannose-binding Lectin protein (MBL) has been suggested to be relevant in the defence against infections in immunosuppressed individuals. In a Swedish adult cohort immunosuppressed from both the underlying disease and from iatrogenic treatments for their underlying disease we investigated the role of MBL in susceptibility to infection.
METHODS: In this cross sectional, prospective study, blood samples obtained from 96 neutropaenic febrile episodes, representing 82 individuals were analysed for single nucleotide polymorphism (SNP) in the MBL2 gene. Concurrent measurement of plasma MBL protein concentrations was also performed for observation of acute response during febrile episodes.
FINDINGS: No association was observed between MBL2 genotype or plasma MBL concentrations, and the type or frequency of infection. Adding to the literature, we found no evidence that viral infections or co-infections with virus and bacteria would be predisposed by MBL deficiency. We further saw no correlation between MBL2 genotype and the risk of fever. However, fever duration in febrile neutropaenic episodes was negatively associated with MBL2 SNP mutations (p<0.05). Patients with MBL2 SNP mutations presented a median febrile duration of 1.8 days compared with 3 days amongst patients with wildtype MBL2 genotype.
INTERPRETATION: We found no clear association between infection, or infection type to MBL2 genotypes or plasma MBL concentration, and add to the reports casting doubts on the benefit of recombinant MBL replacement therapy use during iatrogenic neutropaenia.

Introduction

Mannose-Binding Lectin protein (MBL) is a member of the collectin family of proteins which function as pattern recognition molecules in the innate immune system. MBL recognises and binds to sugar groups present on a wide range of microorganism and plays a significant role as a first line defence against invading pathogens by triggering the complement pathway using MBL-associated serine proteases and possibly functioning as a toll-like receptor co-receptor [1], [2]. Single nucleotide polymorphisms (SNPs) located within the promoter region and exon 1 of MBL2 have been correlated with MBL serum levels [3]. Within exon 1, the wildtype variant is termed ‘A’ and relevant SNP mutations at positions +154 (D variant), +161 (B variant) and +170 (C variant) are collectively denoted by ‘O’. In the promoter regions, three relevant SNP loci have been identified at positions -619 (H/L), −290 (X/Y) and −66(P/Q). In the presence of wildtype ‘A’, HYP, LYP and LXP have been associated with high, intermediate and low MBL levels respectively [3], [4]. The combinatorial effect of SNPs in the promoter and coding region of MBL2 results in variations in MBL concentrations [3], [4]. Whilst constitutional MBL deficiency is unlikely to be clinically relevant in healthy adults [5], this may not be the case in patients with myelosuppression from chemotherapy. Antineoplastic chemotherapy primarily affects the cellular components of the adaptive and innate immune system rendering acellular components of the innate immune system such as MBL potentially important in the role of microbial defense.

Infection episodes are common in patients after induction of chemotherapy during the subsequent neutropaenic phase and account for significant mortality [6]. In 2001, Neth et al. reported a significant increase in the duration and frequency of chemotherapy-induced neutropaenic febrile episodes amongst children with serum MBL deficiency [7]. Subsequent studies in adult patients treated with antineoplastic chemotherapy have yielded conflicting conclusions and metanalyses of data from adult patients are lacking. Low plasma protein levels of MBL has been associated to serious infections related to chemotherapy by Peterslund et al., and Vekemans et al. conclude that while MBL deficiency does not predispose to more frequent or prolonged febrile episodes during myelosuppressive therapy, an association with more severe infections exist [8], [9]. In contrast, Bergmann et al. and Kilpatrick et al. dismiss any strong relationship between low MBL levels and febrile neutropaenia [10], [11]. Furthermore, Lopez et al. and Klostergaard et al. did not find an association between MBL2 genotype and number of infectious episodes following chemotherapy against follicular lymphoma and occurrence of septicaemia, respectively [12], [13].

Comparisons between previous studies are inherently complicated by different distribution of haematological malignancies in the cohorts, different therapies administered and measurement of either serum MBL levels or MBL2 genotype and induction of MBL by inflammation could introduce a temporal sampling bias in studies relying on only protein measurement. Furthermore, association to infection aetiology, apart from unspecified bacteraemia, has yet not been performed. In addition to earlier reports on MBL binding to viral glycoproteins [14], [15], MBL has recently been shown to have protective effects against Ebola virus in an in vivo murine model [16].

As recombinant human MBL has been put through phase I studies [17] based on the suggestion that it is able to decrease infection-related complications in MBL deficient patients with iatrogenic neutropaenia, further study of the role of MBL and other innate constituents in granulocytopaenia are warranted. Here, we sought to study the association of plasma MBL levels and MBL2 coding and promoter region genotypes to infection, type of infection and, febrile episodes in adults with chemotherapy induced neutropaenia.

Materials and Methods

Study population

The study was approved by The Regional Ethical Review Board in Stockholm, permit numbers 2007/1213-31/4 and 2008/1300-32.

During a 26 month period (January 2008 to February 2010), adult patients with haematological disorders at the Karolinska University Hospital, Stockholm were, after informed written consent, included in a cross sectional study where the inclusion criterion was chemotherapy-induced neutropaenia (absolute neutrophile count ≤500/mm3). Patients that developed fever (auricular temperature >38·0°C twice within an hour or ≥38·5°C at one occasion) were sampled within 72 hours from fever onset, whereas patients without fever were sampled upon routine medical appointments during the neutropaenia episode. Whole blood was collected in EDTA-tubes; plasma and blood clots were stored separately at −80°C until use for MBL measurement and genomic DNA extraction, respectively. Additional blood and nasal pharyngeal aspirates (NPA) samples were collected for microbiological testing. C-reactive protein (CRP) concentrations were measured as a clinical routine on the sampling day and acquired from the medical records. Medical records were retrospectively acquired for all included patients. Data was collected from diagnosis of the haematological disorder, through all neutropaenic episodes and ceased upon the patient's recovery, demise or study closure (June 2010), whichever was earlier. Episodes associated with patients migrating into the Stockholm area after initiation of treatment had been excluded from the retrospective, patient-centric analysis but still included in episode-centric analysis pertaining to MBL2 genotypes and the corresponding plasma protein levels. Patients that underwent liver transplants were excluded since MBL is produced in the liver and genotypes may vary between the donor and recipient. In the same manner, patients that had undergone allogeneic haematopoietic stem cell transplantation (HSCT) were also excluded.

MBL2 genotyping

Six SNPs, rs11003125 (L/H allele), rs7096206 (X/Y allele), rs7095891 (P/Q allele), rs5030737 (D variant), rs1800450 (B variant) and rs1800451 (C variant) were investigated. DNA was extracted from the blood clots with QIAamp blood DNA midi kit (Qiagen) according to the manufacturer's protocol and SNP alleles were subsequently determined by sequencing. A nested PCR protocol was designed to selectively amplify the promoter and exon 1 regions of the MBL2 gene. A pair of primers, MBL2_Out_F -5′- TTGCCAGTGGTTTTTGACTC-3′ and MBL2_Out_R-5′-TGCCAGAGAATGAGAGCTGA -3′ was first used to amplify a 1079 bp segment of the MBL2 gene, including the six SNP loci. PCR amplification was performed in a 50 µl reaction consisting 5 µl DNA template, 1× pfu PCR buffer, 200 µM dNTP, 400 nM of each primer and 1·5 U pfu DNA polymerase (Promega Corporation). Amplification was carried out with an initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 1 minute, 65°C for 1 minute and 72°C for 1 minute, and a final extension at 72°C for 7 minutes. PCR products from the above reaction were then used for further amplification prior to sequencing. Two pairs of primers were designed to flank the SNPs in exon 1 and the promoter regions respectively. Primers for the promoter region are MBL2_PROMOTER_F-5′-TTCCTGCCAGAAAGTAGAGAGG-3′ and MBL2_PROMOTER_R-5′- GGATCCTAAGGAGGGGTTCA-3′ whilst primers for the exon 1 region are MBL2_EXON1_F-5′- AGTCACGCAGTGTCACAAGG-3′ and MBL2_EXON1_R-5′- CAGGCAGTTTCCTCTGGAAG-3′. PCR amplification mix for the promoter region consists of 1 µl PCR product from the Out primer, 1× pfu PCR buffer, 200 µM dNTP, 400 nM of each primer, 1·5 U pfu DNA polymerase and water to a final volume of 50 µl. PCR cycling condition begins with an initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 1 minute, 55°C for 1 minute and 72°C for 1 minute, and a final extension step at 72°C for 7 minutes. For the exon 1 region, the PCR amplification mix consists of 1 µl PCR product from the out primer, 1× pfu PCR buffer, 200 µM dNTP, 800 nM of each primer, 1.5 U pfu DNA polymerase and water to a final volume of 50 µl. PCR cycling condition begins with an initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 95°C for 1 minute, 65°C for 1 minute and 72°C for 1 minute, and a final extension step at 72°C for 7 minutes. PCR products from promoter and exon 1 region were then purified with QIAquick gel extraction kit (Qiagen) after separation with 1.5% agarose and sequenced with ABI3730XL after ABI BigDye Terminator v3.1 (Applied Biosystems) reactions. SNP alleles were then determined according to the sequencing chromatograms.

MBL plasma concentrations

MBL plasma concentrations were measured with a commercially available MBL oligomer ELISA kit (Bioporto Diagnostics). The dynamic range for the assay was 0.5 µg/L −40 µg/L and plasma samples were diluted 100 to 1000 times for measurement within this range.

Microbiology

Bacteria were cultured from blood and PCR viral diagnostics for adenovirus, Epstein - Barr virus and cytomegalovirus were performed, as per normal clinical routine, by the local clinical microbiology laboratory, Karolinska University Hospital. Viral diagnostics for BK polyomavirus from blood samples and, respiratory viruses from NPA samples were however, based on PCR methods described in previous studies [18], [19], [20], [21], [22], [23], [24] and Ohrmalm et al [25].

Statistical analysis

Statistical analyses were made with the Prism suite (Graphpad Inc). Fisher's exact test and Mann-Whitney were used for investigating the patients' general characteristics while the chi-squared test was used for determining congruence of SNP prevalence with a reference Danish population and determining Hardy-weinberg equilibrium. Kruskal-wallis test was used for all other analyses where comparison of continuous data for 3 groups or more were required.

Results

A total of 109 episodes of fever during neutropaenia were sampled and 13 were excluded due to insufficient genomic material for genotyping. Thirty-three afebrile neutropaenic episodes were also included as controls. This amounted to 82 febrile neutropaenic patients and 26 afebrile neutropaenic patients (Table 1 and 2). The majority of patients were treated for acute leukaemias and none were diagnosed with invasive fungal infections. Genotype frequencies for the six SNPs investigated, and seven common haplotypes were in Hardy-weinberg equilibrium (Table 3) and were congruent with previous studies conducted amongst healthy Danes [3], [4]. MBL concentrations in our study were also similar to those observed in another chemotherapy-induced neutropaenic cohort [10]. In line with previous studies, mutations in the MBL2 coding region corresponded to a reduction (p<0.001) of plasma MBL concentrations (Table 2). Amongst A/A individuals, mutations in the H/L SNP locus did not result in any significant change in MBL concentrations. X/Y locus mutations correlated with significantly (p<0·001) reduced MBL concentrations and the much less studied P/Q locus contributed to significant (p = 0·0149) concentration reduction as well. Hence, in the presence of functional MBL protein, the impact of the promoter and P/Q SNPs increase in the following order; H/L