The Impact of the NOD2/CARD15 Variant (3020insC) and PSMA6 Polymorphism (-8C>G) on the Development and Outcome of Multiple Myeloma

Introduction Multiple myeloma (MM) is a hematological malignancy characterized by genetic variety. The 3020insC variant of the NOD2/CARD15 gene results in the upregulation of proinflammatory cytokines. Chronic inflammation and abnormal function of the proteasome system may lead to MM development. The polymorphism (-8C>G) in the PSMA6 gene affects proteasome activity. The aim of our study was to analyze the possible relationship of NOD/CARD15 and PSMA6 genes with the risk of development and outcome of MM, as well as the sensitivity to bortezomib (proteasome inhibitor) in cell cultures derived from MM patients. Objects and Methods. Genomic DNA from 100 newly diagnosed MM patients and 100 healthy blood donors was analyzed by methods such as PCR-RFLP (for PSMA6 genotyping) and automated DNA sequencing (for NOD2/CARD15 genotyping). In a subgroup of 50 MM patients, nucleated bone marrow cells were treated with bortezomib in vitro. Results Patients with PSMA6 CG+GG genotypes had higher chances for progressive disease (OR = 5.0, 95% CI 1.07-23.16, p = 0.05), shorter overall survival taking into account the type of treatment (p = 0.039), and increased risk of death due to MM at the level of tendency (OR = 4.74, 95% CI 1.02-21.97, p = 0.06). The presence of NOD2/CARD15 3020insC decreased the risk of renal dysfunction in MM (OR = 0.23, 95% CI 0.07-0.74, p = 0.009). The analyzed changes in NOD2/CARD15 and PSMA6 genes did not impact the MM risk. In an in vitro study, bortezomib increased the number of apoptotic cells at 8 nM and 12 nM between wild-type and 3030insC variants of NOD2/CARD15 (p = 0.018 and p = 0.03, respectively). Conclusion The presented results suggest a possible impact of PSMA6 CG+GG genotypes on the MM outcome and the association of the NOD2/CARD15 variant with bortezomib in vitro sensitivity.


Introduction
Multiple myeloma (MM) is a hematological, heterogeneous malignancy characterized by infiltration of bone marrow by clonal plasma cells [1]. Inflammatory mediators, such as nuclear factor κB (NF-κB), are critical in the malignant transformation [2,3]. Chronic inflammation with elevated NF-κB can support the development of hematological malignancies, including MM [4]. Activation of this pathway is enhanced by nucleotide oligomerization domain 2 (NOD2 also known as CARD15) [5]. This protein is encoded by the NOD2/-CARD15 gene (locus 16q12.1), is a component of the host defence against intracellular pathogens, and plays an important role in immune system function. The NOD2/CARD15 gene consists of 18 exons, and its expression is observed in monocytes, macrophages, granulocytes, and some epithelial cells [5,6]. The most common variant in the NOD2/CARD15 gene is present in the form of 3020insC, which leads to a stop codon in exon 11. The result of a nonsense change is the production of truncated NOD2 protein with the last 33 amino acids missing (Leu1007fsinsC) [5]. The variant leads to loss-of-gene function, and truncated protein has a reduced ability to recognize bacterial muramyl dipeptide and consequently to stimulate NF-κB response [5,7]. On the other hand, the absence of wild-type NOD2 protein results in the upregulation of proinflammatory cytokines [8]. The 3020insC variant of NOD2/CARD15 also predisposes cells to many types of common cancers, mainly solid tumors-for example, ovarian cancer, breast cancer, and colorectal cancer [7,9,10].
The processes like apoptosis, proliferation, and inflammation are controlled in a nondirective way by the proteasome system [11]. It is a multicatalytic proteinase complex. The 20S proteasome is composed of seven α-subunits and ten β-subunits. The human proteasome subunit α6 (or p27K) is encoded by the PSMA6 gene (locus 14q13), which contains single-nucleotide polymorphism (SNP) in 5 ′ -UTR (rs10489900). This SNP is caused by substitution (-8C>G) where nucleotide with cytosine (C allele) is replaced by guanine (G allele). The G allele is associated with a higher transcription rate of the PSMA6 gene in human B cells in comparison to the C allele [12]. The proteasome-dependent pathway and higher expression of the PSMA6 gene may activate NF-κB, which leads to inflammation and transformation to MM [13].
The proteasome system consists of the 26S proteasome (including two subunits of 20S and 19S) and immunoproteasome [14]. The 26S proteasome is involved in the degradation of proteins regulating the cell cycle, cell migration, proliferation, angiogenesis, and apoptosis [15]. These proteins play a role in malignant transformation. Therefore, proteasome inhibitors preventing angiogenesis and cell migration and decreasing inflammatory response have been developed and tested for antitumor response [14,15]. In MM patients, significant improvements in survival have been obtained, among others, by the introduction of bortezomib, a 26S proteasome inhibitor, as well as an anti-inflammatory agent-thalidomide [16]. Inhibition of the proteasome pathway in cancer cells leads to apoptosis via the activation of unfolded or abnormal protein response [17].
Taking the above into account, we hypothesized that variants in the NOD2/CARD15 gene and PMSA6 polymorphism may affect the development and outcome of MM. In this paper, we present an analysis for the studied genetic variants individually and in different combinations to evaluate the role of these genes in the MM susceptibility, survival, and response to treatment with thalidomide and/or bortezomib. Furthermore, the aim of our research was to analyze the studied DNA changes in the sensitivity prediction in cell cultures derived from MM patients. To our knowledge, the presented results were not previously published by other authors. Control samples were made of peripheral blood obtained from 100 healthy blood donors (50 males/50 females, with mean age 34.4 years) attending the Regional Blood Donation and Blood Treatment Center in Kielce, Poland.

Material and Methods
The general characteristics of MM patients are shown in Table 1. The inclusion and exclusion criteria are described in Table 2. Therapeutic induction regimens consisted of thalidomide and/or bortezomib combined with steroids and/or cyclophosphamide. 37 MM patients underwent autologous hematopoietic stem cell transplantation (auto-HSCT). Response to treatment was evaluated according to the International Myeloma Working Group guidelines and classified as stringent complete remission (sCR), complete response (CR), very good partial response (VGPR), partial response (PR), minimal response (MR), stable disease (SD), or progressive disease (PD) as described elsewhere [18,19]. Overall survival (OS) encompassed time from diagnosis until relapse, progression, death due to a tumor effect, or the last follow-up and time from diagnosis until death by any cause or the last follow-up. The median follow-up time of MM patients enrolled in the study was 18 months. Progression-free survival (PFS) was estimated as the time elapsed between treat-ment initiation and tumor progression or death from any cause [20].
Peripheral blood was used for DNA isolation and studied genetic variant determination.
Cell cultures were established from bone marrow aspirates to carry out the research associated with cIg-FISH (n = 100) and bortezomib treatment (n = 50). In an in vitro bortezomib study, MM patients without chromosomal aberrations were included.
2.2. DNA Isolation. DNA isolation from peripheral blood was performed using a commercial kit (Qiagen, Germany) according to the manufacturer's procedure. The concentration and quality of DNA were checked using a NanoDrop device (Thermo Fisher Scientific, USA).

PSMA6
Genotyping. For the analysis of the PSMA6 polymorphism, the PCR restriction fragment length polymorphism (PCR-RFLP) method was applied according to the validated protocol of Bachmann et al. [21]. A PSMA6 gene fragment length of 100 bp was amplified by PCR using the following primers: Each PCR mixture (25 μl) contained 100 ng genomic DNA and PCR buffer (Clontech), dNTP mixture (0.25 mM), HD polymerase (Clontech), and primers (10 μM of each). The touchdown PCR method was used. The mixture was heated at 95°C for 5 min and underwent 14 cycles of amplification: denaturation at 95°C for 30 s, annealing at 64.5°C for 20 s (-0.5°C/per cycle), and elongation at 72°C for 20 s. After 14 cycles, the mixture underwent 20 cycles with a constant temperature of 57.5°C. The denaturation and elongation temperatures and times were the same as the above. The final elongation takes 5 min at 72°C. The PCR was performed in the Applied Biosystems 9700 Thermal Cycler.
The PCR product was digested with RsaI (Thermo Fisher Scientific) for 16 hours at 37°C producing two fragments of 50 bp or one fragment of 100 bp for the presence of the G or C allele, respectively. RFLP products were analyzed on 3% agarose gel and stained with SimplySafe (EURx, Poland) and visualized in G:Box (Syngene, Great Britain) ( Figure 1). An independent PCR analysis was carried out for each sample.  HD polymerase (0.31 U) (Clontech), and primers (10 μM of each). The mixture was heated at 94°C for 5 min and underwent 35 cycles of amplification: denaturation at 98°C for 15 s, annealing at 60°C for 10 s, and elongation at 72°C for 20 s. The final elongation takes 5 min at 72°C. The PCR was performed in the Applied Biosystems 9700 Thermal Cycler using the HD Advantage polymerase (Clontech). Sequencing PCR was performed with the use of the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems) in a thermal cycler (as described previously). The sequencing PCR product was purified by the use of an exterminator kit (A&A Biotechnology). The sequencing run module was StdSeq50_ POP7 in genetic analyzer 3130 (Applied Biosystems). The results were analyzed by the use of Applied Biosystems software ( Figure 2).

Cytogenetic Analyses.
Abnormalities essential for MM, such as del(17p13.1) and IgHV gene rearrangements-t(4;14) and t(14;16)-were tested by simultaneous staining of cytoplasmic immunoglobulin and FISH (cIg-FISH) according to Ross et al.'s recommendations and were identified according to the previously described protocol with modifications [20-24].
2.6. Bortezomib In Vitro Treatment. Bone marrow aspirates (n = 50) (mean number of plasma cells-31:31% ± 20:69) were used to establish cell cultures as described by Zmorzynski et al. [20]. The number of apoptotic, necrotic, and viable cells was determined by means of the Annexin V-Cy3 Apoptosis Detection Kit according to the manufacturer's protocol (Sigma-Aldrich, USA) and following the methods of Zmorzynski et al. [24].

Statistical Analysis.
Laboratory values of MM patients with polymorphisms were compared using an independent t-test for continuous variables and the chi-square test for categorical variables. The association of the studied polymorphisms with prognostic factors was evaluated using the chi-square test or Fisher's exact test (when one expected value was <5). The quantitative data was shown as frequency or percentage. Deviation of genotype frequencies in controls and cases from the Hardy-Weinberg equilibrium (HWE) was assessed by the chi-squared test with Yates's correction for the groups with less than five patients [25]. For 95% confidence interval (CI), we assumed p = 0:05 and χ 2 = 3:84; therefore, if χ 2 ≤ 3:84 and the corresponding p ≥ 0:05, then the population is in HWE. The logistic regression was used to evaluate the fold risk of MM. The Cox proportional hazard model was used for univariate and multivariate analyses of OS and PFS. Variables of the international staging system (ISS), auto-HSCT, and genotypes (without combinations) were included in multivariate analysis. The Kaplan-Meier method and the log-rank test were used for survival analysis. The statistical power of the study was calculated according to Bacchetti and Leung [26]. We assumed a 5% error of inference and the related level of significance p < 0:05 pointing to the existence of statistically significant differences. Statistical analyses were performed using the Statistica ver. 12.5 (StatSoft) software.   T  T  G  A  A  A  G  187  188  190  189  191 192 193  194  195 196  197  198  199  200 201 202  204 G 203

Results
The presented study included 100 MM patients, 53 males and 47 females, with a median age of 65.32 years. Genotyping was successful in all the individuals investigated within the study. The HWE test confirmed that the genotypic frequencies of PSMA6 and NOD2/CARD15 genes for healthy individuals (controls) and MM patients were balanced ( Table 3). The allelic frequencies of the PSMA6 or NOD2/CARD15 gene between study and control groups were statistically insignificant ( Table 4). The studied PSMA6 and NOD2/CARD15 variants did not impact on the risk of MM ( Table 5). The CG and GG genotypes of the PSMA6 gene were analyzed together, because the number of GG cases was very low (Table 3). Moreover, we analyzed the effect of both changes-3020insC of the NOD2/CARD15 gene and CG+GG genotypes of the PSMA6 polymorphism-on the risk of MM, which was statistically insignificant (Table 5). In the case of the 3020insC variant of the NOD2/CARD15 gene, only heterozygotes were observed.
A univariate Cox analysis revealed that patients at stage III according to ISS had a 1.7-fold increased risk of death (p = 0:003) ( Table 6). Similar findings were observed in the case of disease relapse or progression in MM patients at stage III (HR = 1:68, p < 0:001) and without auto-HSCT (HR = 2:54, p = 0:002) ( Table 6). Moreover, the univariate Cox analysis did not show an increased risk of death or disease relapse/progression in MM patients with the PSMA6 polymorphism and/or NOD2/CARD15 variant. The multivariate Cox regression analysis confirmed that patients without auto-HSCT or at stage III according to ISS had an increased risk of death or disease relapse/progression, respectively ( Table 7).
The analysis of the response rate in MM showed that patients at stage III or without auto-HSCT had an increased chance of progressive disease (PD) ( Table 8). Moreover, patients with CG+GG genotypes of PSMA6 had higher chances for PD ( Table 8). The statistical power of this association was 0.63. The CG+GG genotypes had an increased risk of death due to MM at the level of tendency (OR = 4:74, 95% CI 1.02-21.97, p = 0:06).
Furthermore, we analyzed the potential relationships between clinical and laboratory results and selected genotypes. We found that the NOD2/CARD15 variant decreased the risk of renal dysfunction in MM patients (OR = 0:23, 95% CI 0.07-0.74, p = 0:009). We did not confirm any significant correlations among the studied variants of NOD2/-CARD15 and PSMA6 genes regarding clinical parameters ( Table 9). The odds ratio was used to analyze the relationship of the PSMA6 polymorphism and NOD2/CARD15 variant with chromosomal aberrations in MM patients. The results were statistically insignificant for PSMA6 CG+GG      We analyzed (by ANOVA) the association between the studied genotypes and survival of MM patients. Without taking into account the treatment, we did not find statistically shorter OS and PFS in patients with the wild-type NOD2/-CARD15 or PSMA6 CC genotype in comparison to those with the 3020insC variant of NOD2/CARD15 or PSMA6 CG+GG genotypes (Table 9). In the log-rank test (Figures 3  and 4) without taking into account the type of treatment, the tendency for shorter PFS in patients with CG+GG genotypes was observed ( Figure 4). Furthermore, a log-rank analysis taking into account the studied changes and the types of treatment (thalidomide versus bortezomib versus both-thalidomide and bortezomib) was performed. The NOD2/-CARD15 3020insC variant did not impact the type of treatment versus OS (p = 0:213) or PFS (p = 0:122). Similar results were observed in the case of the CC genotype of the PSMA6 gene (OS (p = 0:182) and PFS (p = 0:095)). Individuals with CG+GG genotypes treated with bortezomib     In in vitro studies, bortezomib increased the number of apoptotic and necrotic cells in all the studied genotypes. The only statistically significant differences were observed in the number of apoptotic cells at concentrations of bortezomib at 8 nM and 12 nM between wild-type and 3020insC variants of NOD2/CARD15 (Table 10).

Discussion
To our knowledge, this is the first study elucidating the correlation of the PSMA6 polymorphism and NOD2/CARD15 variant with the risk and the outcome of MM, as well as the response to bortezomib under in vitro conditions.
Little is known about the role of the PSMA6 -8C>G polymorphism in cancer development, including MM. Most of the research was focused on the association of this SNP with the risk of cardiovascular disease and diabetes mellitus [27,28]. Our findings suggest that CG+GG genotypes of the PSMA6 gene might be associated with the response rate of MM patients and shorter OS taking into account the type of treatment. The G allele of this SNP leads to higher promoter activity and elevated PSMA6 gene expression in human B cells in comparison to the C allele [12]. Higher pro-teasome activity, as a result of the GG genotype, may lead to degradation of misfolded or unfit antiapoptotic proteins [29]. Bachmann and coworkers in 116 MM patients showed shorter median survival time for individuals with the GG genotype [21]. Moreover, they revealed in Cox multivariate analysis that the G allele was an independent prognostic factor for MM patients [21]. In our Cox multivariate analysis, we did not confirm the association of the G allele with the MM outcome. However, in the log-rank test, we observed higher chances of progression in patients with CG+GG genotypes. Additionally, taking into account the type of treatment, we found shorter OS in individuals with CG+GG genotypes treated with bortezomib/bortezomib +thalidomide in comparison to those (with CG+GG genotypes too) treated with thalidomide. It has a different mechanism of action in comparison to bortezomib. Thalidomide, as an immunomodulatory drug, inhibits the NF-κB pathway and provides better survival of MM patients [30]. The inflammation development through activation of the NF-κB pathway may lead to malignant transformation, as well as disease progression [13].
The α6 subunit, encoded by the PSMA6 gene, is present in the core of the 20S proteasome. It belongs to proteasome-interacting proteins and among others with the β5 subunit plays a role as an auxiliary factor. Suppression of the proteasome leads to endoplasmic reticulum stress and apoptosis [31]. Inhibition of the proteasome using β5 subunit blockers like bortezomib results in cell cycle arrest and induction of apoptosis in MM cells [32]. However, in our study, we did not observe the relationship between the     The change in the NOD2/CARD15 gene in the form of C insertion in a region encoding the ligand recognition domain (exon 11) is described as polymorphism (rs2066847) or mutation [5,9,10,33,34]. The difference is due to the fact that in cancer research, it is called mutation and in a noncancer field, the term polymorphism is used. The current guidelines of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology recommend the use of the neutral term "variant" only [35]. The 3020insC variant of the NOD2/-CARD15 gene is implicated in the development of chronic inflammation and increased risk of solid tumors such as colorectal, gastric, breast, and lung cancer, as well as non-Hodgkin's lymphoma [10,[36][37][38][39]. Kurzawski et al. found a statistically significant difference in mutated NOD2/CARD15 allele distribution in 250 nonhereditary nonpolyposis colorectal cancer patients in comparison to healthy individuals [36]. In our study, we did not observe differences in NOD2/CARD15 wild-type/3020insC allele distribution between MM patients and the control group. The frequency of these alleles in our study populations was in HWE. Moreover, we did not find a statistically significant impact of the NOD2/CARD15 variant on OS and PFS of MM patients. This result is consistent with the observation of Huszno and coworkers. In their study in breast cancer patients, they found no difference in OS between 3020insC variant carriers and wild-type homozygotes of the NOD2/CARD15 gene [9].
Furthermore, we found that the NOD2/CARD15 variant decreased the risk of renal dysfunction in MM patients, which was not previously reported by other researchers. In an animal model, Stroo and coworkers observed no difference in renal injury between wild-type Nod2 doubleknockout mice with chronic kidney disease [40]. They concluded that Nod2 does not play an important role in the development of renal damage [40]. On the other hand, Kruger et al. observed an unfavorable effect of the NOD2/-CARD15 wild type on the outcome of graft survival in 352 patients receiving their first renal transplant [41]. Moreover, they took into account two additional SNPs (SNP8-R702W and SNP12-G908R) present in the NOD2/CARD15 gene, which are associated with increased susceptibility to Crohn's disease [41]. It is possible that the NOD2/CARD15 variant showed only a minor effect and cooccurrence of these SNPs may affect or modify the function of NOD2 protein. In our further research, it would be worth extending the analysis by these two SNPs.
In our in vitro study, the 3020insC variant of the NOD2/CARD15 gene was associated with statistically significant lower levels of apoptotic cells at 8 nM and 12 nM of bortezomib. The loss of function of the NOD2/CARD15 gene in MM leads to apoptosis inhibition. It is known that overexpression of NOD2/CARD15 enhances apoptosis via caspase-9 [42]. It would be desirable to examine the

12
BioMed Research International NOD2/CARD15 gene expression in plasma cells after in vitro bortezomib treatment.
There are some limitations of our study associated with the number of MM patients recruited to the study, as well as the used methods.
The sample size was relatively small, in part due to the low incidence of the disease. We have collected detailed clinical data for 100 MM patients. Some patients were not included into this analysis, because they are still in followup and their clinical data are incomplete. However, in our research, the number of 100 MM patients is enough for most analyses. Some of them were not possible as a result of low frequency of genotypes. Further analysis on a larger cohort can help better understand the significance of the studied changes in the pathobiology of MM, especially in the case of disease outcome.
In our in vitro study, we used RPMI media with 10% FCS. Currently, AIM-V media are used instead of RMPI and FCS as they induce apoptosis of primary B cells [43]. In our control samples without bortezomib, spontaneous apoptosis was observed, and therefore, in our further research, apoptosis evaluation will be done using serum-free AIM-V medium. Moreover, for the evaluation of apoptosis, fluorescent microscopy instead of flow cytometry-based apoptosis detection (FACS) was used. FACS analysis is more reliable and quantitative for apo-ptosis evaluation. Unfortunately, during the experiment time, the flow cytometry method was not available for us and retrospective analysis of apoptosis is not possible. The set used for apoptosis and necrosis detection was dedicated and validated to fluorescent microscopy.
In this paper, we analyzed, for the first time, the significance of the PSMA6 polymorphism and NOD2/CARD15 variant in the survival and response to therapy of MM patients. In conclusion, these data underscore the possible relationship between PSMA6 CG+GG genotypes and response rate of MM patients, as well as shorter OS taking into account the type of treatment. Moreover, we observed an association of the NOD2/CARD15 variant with the lower risk of renal dysfunction in the study group and lower apoptosis levels in cell culture with bortezomib.

Data Availability
The clinical data used to support the findings of this study are available from the corresponding author upon request.

Conflicts of Interest
The authors declare that they have no conflicts of interest.