Association Between Mannose-binding Lectin And Infection In Multiple Myeloma Patients During Induction Chemotherapy

BackgroundMultiple myeloma(MM) is a malignant clonal plasma cell disorder. The main characteristic is the abnormal proliferation of clonal plasma cells and the secretion of monoclonal protein (M protein), including immunoglobulin or light chain. However, a little proportion of MM patients do not secrete M protein. The accumulation of clonal plasma cells and M protein resμlt in the failure of organs and occurrence of clinical symptoms. The most common symptoms are bone pain, anemia, renal impairment and infection. Infection is one of the most common symptoms in the newly diagnosed MM patients, and one of the most serious complications during the treatment, and also one of the main causes of death in MM patients. Studies showed that: about10%of newly diagnosed MM patients died within60days after induction therapy and approximately50%of the patients died from infection. The main reasons for patients suffered from infection are the abnormal proliferation of neoplastic plasma cells in the bone marrow, the secretion of monoclonal immunoglobulin or κ/γ light chain, and the inhibition of the synthesis of normal immunoglobulin, which lead to the defection of humoral immunity. In addition, the decrease of T/B cells and neutrophils, the disfunction of T/B cells, chemotherapy-related damages, and the application of adrenal cortical hormone, also lead to a further disfunction of the immune systems and an increase of the risk of infection in MM patients.Mannose-binding lectin (MBL) is one of the complement system of innate immune system. It consists of several identical polypeptide chains, and each chain has four parts:N-terminal cysteine-rich region (N-TS), collagen-like region (CLR), a-helical coiled hinge region (neck region)(HCHR), C-terminal Ca2+-dependent carbohydrate recognition domain (CRD). CRD can recognize the sugars on the surface of various microorganisms, including N-acetyl glucosamine, N-acetyl mannosamine, D-mannose, L-fucose, etc. Three identical polypeptide chains form a subunit, and two or more subunits form an oligomer (e.g. dimers, hexamers, etc.), but only tetramers or more are the effective forms.The plasma level of MBL, which is mainly synthesized by the liver cells, is decided by genotype. Studies have shown that the plasma level of MBL is mainly influenced by the polymorphism of-619(H/L),-290(X/Y),-66(P/Q) in promoter region and codon52(A/D), codon54(A/B), codon57(A/C) in exon1of MBL gene. The variation in the promoter region may influence the synthesis of MBL in the transcriptional level. The variations in exon1may cause the normal amino acid in collagen-like region replaced (such as, arginine�'cysteine, glycine�'aspartic acid, glutamic acid�'glycine), and result in a low plasma level of MBL through a destruction of the structure of the polypeptide chain.MBL resist the invasion of pathogens by activating the complement system, promoting the complement-independent opsonophagocytosis and modulating the inflammation through the recognition and binding of the special sugars on the surface of microorganisms. So the plasma level is very important. In recent years, several studies abroad showed that patients with tumor who had a higher plasma level of MBL or carry the genotypes of high MBL expression related to lower incidence of infections, indicating that MBL may mediate the resistance to infection during chemotherapy for patients with malignancy. However, other studies showed that there was no association between polymorphism of MBL gene/plasma levels of MBL and the incidence of infection during chemotherapy. The convincing conclusion has not been reached yet. In decades, there were only two studies from the same center that investigated the relationship between MBL and infection in MM patients during chemotherapy. They found that MM patients with AA genotype in exon1had lower incidence rate of severe infection comparing with AO/OO patients(B/C/D variation were all called O variation) during autologous hematopoietic stem cell transplantation. There isn’t any related research in China up to now.ObjectiveThe aim of this study is to explore whether MBL-deficient are the risk factors for infection in MM patients during induction chemotherapy or not.Methods1Patients selectionAccording to the2011Guidelines for Multiple Myeloma diagnostic and treatment in China, the newly diagnosed patients with MM who were treated in our hospital from January1,2012to September30,2013were selected. Finally, a total of60patients were enrolled.2Experimental MethodsWe collected the peripheral blood DNA and plasma samples of60untreated MM patients before the first chemotherapy began. Then, we detected the variations of MBL2gene and the plasma MBL level by polymerase chain reaction sequencing and enzyme-linked immunosorbent assay, respectively.3The contents of the study First, we compared the frequency of gene and genotype between MM patients and normal persons; Secondly, we analyzed the association between variations in MBL2gene and the plasma levels of MBL. Thirdly, we analyzed the relationship between the variations of MBL2gene and frequency of infection in MM patients during the first two induction chemotherapy through three aspects. Finally, we analyzed the association between the plasma levels of MBL and infection in newly diagnosed patients with MM during induction chemotherapy.4Statistical analysisStatistical analysis was performed using SPSS for Windows, version17.0. Firstly, we used Fisher’s exact test, Mann-Whitney U-test, t test to compare the clinical data between different groups in the gene and plasma levels. Secondly, we analyzed the relationship between the variations of MBL2gene and plasma levels of MBL and infection in patients with MM during the first two induction chemotherapy through Mann-Whitney U-test and Kruskal-Wallis H-test, respectively. All P values are two-way. A value of P<0.05was considered statistically significant.Result1Polymorphism of MBL2gene in MM patientsMBL2gene polymorphisms were tested among47patients, only four sites, including H/L, X/Y, P/Q and A/B, were detected the variation. The variation of A/D and A/C cann’t be found.Genotype/gene frequencies for the four SNPs investigated were in Hardy-weinberg equilibrium, and Genotype/gene frequencies for all six SNPs were congruent with PubMed database conducted among healthy Chinese.Only five haplotypes (HYPA, LYPA, LYQA, LXPA, LYPB) and13genotypes (HYPA/HYPA, LYPA/HYPA, LYQA/HYPA, LXPA/HYPA, LYQA/LYQA, LYPA/LXPA, LYQA/LXPA, LXPA/LXPA, LYPB/HYPA, LYPA/LYPB, LYQA/LYPB, LYPB/LXPA, LYPB/LYPB)were found because of the linkage disequilibrium between them.2Polymorphism of MBL2gene and plasma level of MBL44patients were both tested MBL2gene polymorphisms and plasma level were both tested at the same time. We analyzed the association between plasma levels of MBL and the polymorphism of sites within promotor region and exonl in turn. The results showed: A/B site of exonl and H/L site of promotor region had a significant influence on the plasma concentration of MBL for all patients (P=0.000, P=0.000). The median plasma level of MBL in AA patients>AB genotype>BB genotype(2396.68ng/ml vs536.06ng/ml vs73.22ng/ml), and so was the H/L site:HH genotype>HL genotype>LL genotype (2669.62ng/ml>1829.89ng/ml>509.35ng/ml). X/Y、P/Q sites had no influence on plasma concentration of MBL(P>0.05).Because of the influence of A/B and H/L sites on plasma concentration, we analyzed the relationship between genotype and plasma concentration. The results showed that patients with A/A genotype had a higher median MBL plasma levelthan the ones with A/B and B/B genotype. From the above, we knew that A/B had a greatest influence on MBL plasma level. So we divided patients into AA and AB/BB groups and analyzed the influence of other sites on AA or AB/BB patients. Finally, we found that H/L site had a significant influence on both AA or AB/BB patients(P=0.002, P=0.003), while Y/X site only influenced AA patients(P=0.000). There were no association between P/Q sites and the plasma level of MBL(P>0.05).In summary, considering the influence of both H/L and A/B sites on plasma MBL levels, we divided patients into HA/HA, LA/HA, LA/LA, LB/HA, LA/LB, LB/LB genotypes, and analyzed the relationship between plasma levels of MBL and genotype. The result showed that the difference between different genotype was significant(P=0.000), and the median plasma MBL levels in HA/HA patients>LA/HA type>LA/LA type>LB/HA type>LA/LB type>LB/LB type (2783.15ng/ml vs2396.68ng/ml vs1536.06ng/ml vs662.77ng/ml vs228.87ng/ml vs73.22ng/ml).3Association between MBL2gene polymorphism and infection in newly diagnosed MM patients during induction chemotherapyThe proportion of patients suffered from fever of unknown,clinical infection,pneumonia and infection in AA patients is lower than that in AB/BB patients (12.12%vs28.58,36.36%vs42.86%,48.48%vs71.43%), but it didn’t reach the significant difference(P>0.05). However, patients with AA genotype had a shorter duration of fever than the ones with AB/BB genotype (median duration of fever:2days vs8days, P=0.001).A/B and H/L sites had a great influence on plasma level of MBL, so we divided patients into HA/HA, LA/HA, LA/LA, LB/HA, LA/LB, LB/LB, and analyzed he cumulative incidence of fever of unknown, clinical infection, pneumonia and infection in patients with different genotype. The results showed no significant difference(P>0.05). And the accumulated duration of fever in fever patients with different genotypes had no significant difference (P>0.05), too.4Association between the plasma level of MBL and infection in newly diagnosed MM patients during induction chemotherapyThe median plasma level of MBL in the patients with no infection were significantly higher than patients with infections (2362.44ng/ml vs1576.13ng/ml, P=0.019). And the patients with twice or more infections had a lower median plasma level of MBL than patients with1or0infection (163.45ng/ml vs1796.48ng/ml vs2362.44ng/ml, P=0.008).We divided the patients into two group using1000ng/ml as the boundary. The cumulative incidences of pneumonia and infection in the patients with the plasma MBL level below1000ng/ml was higher than the ones above1000ng/ml (median incidences:1VS0, P=0.014,1VS0, P=0.001). And the cumulative duration of fever was also longer than the latter (median days:7.5days vs2days, P=0.006)Conclusion1The genotype/gene frequences of the six sites within promotor region and exonl among MM patients are congruent with PubMed database conducted amongst han Chinese.2The influence of the four sites detected on MBL plasma level is in sequence of A/B type>H/L type> X/Y type, and the plasma level of MBL in patients with variation in P/Q is not different.3The variation of A/B site is related to the occurrence of infection for untreated MM patients. MM patients with AA genotype have a shorter accumulate duration of fever and has a tendency of lower accumulate occurrence of infections during induction chemotherapy.4MM patients with high plasma levels of MBL had a lower occurrence of infections and a shorter cumulative duration of fever in patients with MM during chemotherapy.