Introduction
Multiple myeloma (MM) is a hematological malignancy that occurs when abnormal plasma cells grow uncontrollably and invade the bone marrow (BM) (1). The average age at diagnosis is 60 years old (2). MM accounts for 1% of all malignancies and 10% of all hematological cancers (3). The incidence of MM is highest in the African population, whereas the lowest rates are observed in the Asian and Mediterranean populations (4). Although advancements in MM treatment have raised the 5-year survival rate to almost 50%, MM is still regarded as incurable. Although the exact cause of MM remains unknown, in previous studies, age, male gender, obesity, exposure to ionizing radiation, and a history of monoclonal gammopathy of undetermined significance (MGUS) have been mentioned as risk factors (5). Many studies have shown that genetic factors play an essential role in the etiology of MM. Genetic factors, particularly those pertaining to immune response genes, increase the likelihood of MM development.
Leptin (LEP), a hormone, is primarily produced by white adipose cells in the body. By affecting energy expenditure and food intake, it has a significant effect on maintaining body weight homeostasis (6). In addition, LEP has various other functions in the body, such as influencing insulin resistance, cell proliferation, oxidative stress, cancer cell inflammation, apoptosis, and immune regulation (7). LEP is found in human peripheral blood, cord blood, and BM blood. Various studies have shown that high levels of LEP may affect the onset and progression of many malignancies. LEP exerts its biological effect by binding to and activating leptin receptors (LEPR). The LEP gene is on chromosome 7q31.3. The leptin receptor encoded by the LEPR gene is a member of the class 1 cytokine receptor family. LEPR plays a role in the pathogenesis of many malignant cancers (8). In vitro and in vivo studies have demonstrated the critical role of LEP/LEPR signaling in the growth, maturation, and functionality of hematopoietic progenitor cells and mature blood cells (9). The LEP and LEPR genes have single-nucleotide polymorphisms (SNPs). The variant -2548G/A (rs7799039) is situated in the promoter region of the LEP gene, whereas the variant 668A/G (rs1137101) of the LEPR gene is located in the exon 6 region of the LEPR gene (10).
Based on this information, in this study, we evaluated whether LEP-2548G/A and LEPR 668 A/G variants cause susceptibility in patients with MM. In addition, the methylation of CpG sites located at -31 and -51 nucleotides (nt) according to the transcription start region of the LEP gene was examined.
Methods
Study population
Nineteen patients (7 females and 12 males, median age: 60 years) diagnosed with MM were included in the study. Staging was assigned according to the Durie and Salmon and the International Staging System criteria (11). The clinical characteristics and treatment protocols of the patients were examined. The 2-year progression-free survival (PFS) and 2-year overall survival (OS) were evaluated according to prognostic factors. The healthy control group consisted of 93 healthy individuals (49 females, 44 males, median age: 45 years). The control group was selected from volunteers without any malignancies or chronic diseases. All participants received written explanations of the study objectives and methods.
The Local Ethics Committee approved all procedures involving human subjects during this study, which was conducted in accordance with the Declaration of Helsinki’s criteria.
The study was approved by the İstanbul University, İstanbul Faculty of Medicine Local Ethical Committee (approval number: 62734, date: 29/05/2020).
Genotyping
Peripheral blood was used to extract DNA samples from the groups. The LEP-2548G/A and LEPR 668A/G variants were analyzed using the polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). The primer sequence used for LEP-2548G/A F:5’-TTTCCTGTAATTTTCCCGTGAG-3’ and R:5’-AAAGCAAAGACAGGCATA AAAA- 3’, the primer sequence for LEPR 668A/G was F: 5’-GCCTAATCCAGTATTTTATATCTG-3’ R: 5’GCCACTCTTAATACCCCCAGT. HhaI restriction enzyme for LEP-2548G/A and MspI enzyme for LEPR 668 A/G were used (12,13). LEP gene -31 and -51 nt methylation was performed using the methylation-specific PCR (MS-PCR) method previously described by García-Cardona et al. (14).
Statistical Analysis
SPSS, version 20.0 for Windows (SPSS, Inc., Chicago, IL, USA) was employed to examine all data. Using Pearson’s c2 analysis, categorical data were evaluated. In addition, the 95% confidence interval (CI) and odds ratio (OR) were computed. OR (95% CI) was age- and sex- adjusted. The log-rank test was used to compare the patient group’s 2-year PFS and OS based on prognostic variables. The threshold for statistical significance was set at a p-value of less than 0.05 in all two-tailed analyses.
Results
LEP-2548G/A, LEPR 668 A/G variants, and -31 and -51 nt of the LEP gene were examined in patients with MM and controls. The clinical characteristics and treatment regimens of the groups are listed in Table 1.
Table 2 summarizes the distributions of genotypes for these variants in groups. LEP-2548G/A and LEPR-668A/G genotype distributions did not differ significantly between the patient and control groups (p>0.05). The methylation status of the -31 and -51 nt regions of the LEP gene significantly differed between patients and controls. There was no methylation in the -31 and -51 nt regions of the LEP gene in the patient group compared with the control group (p=0.051 and p=0.001, respectively).
The patient group’s 2-year PFS and 2-year OS were compared according to prognostic factors. The results are shown in Table 3. The mean 2-year PFS was 22 months, and the mean 2-year OS was 15.2 months. -31 nt methylation was unchanged in 15 patients (78.94%). PFS and OS were higher in these patients than in others (p=0.019 and p=0.048).
In multivariate analysis, the methylated/unmethylated ratio at -31 nt methylation was associated with a poor prognosis (p=0.020) (Table 4).
Discussion
Epidemiological and family studies have determined that the risk of MM and its precursor disease, MGUS, is associated with genetic predisposition (15). One of the known and confirmed risk factors for MM and MGUS is family history. In first-degree relatives of patients with MM, the risk of MM increases two to four times, and the risk of MGUS increases two to three times (16). The initiation and development of MM are accompanied by genetic alterations, such as chromosomal translocations, and structural variants, such as deletions, duplications, and insertions. Deleterious single-nucleotide variations are also frequently observed. These may result in the progression of the illness and treatment resistance (17). Some SNPs have also been shown to play a role in MM susceptibility.
The primary element of the BM microenvironment is crucial for the emergence and development of MM. The BM adipose tissue, which constitutes 50-70% of the BM volume (18). Adipose tissue functions as an endocrine organ that contributes to the release of certain adipocytokines in addition to serving as a store of energy (19). Some studies have shown that changing levels of adipokines secreted from fat tissue play a role in the development of various types of cancer (20). LEP also plays an important role in energy balance and metabolism functions as well as receptors related to adaptive immunity, angiogenesis, and the hematopoietic process (21). LEP levels increase in people with excess fatty tissue that are at increased risk of cancer. This suggests that LEP may play a role in the development of cancer (22). According to certain research examining LEP’s impact on hematological cancers, LEP somewhat promotes myeloma cell line proliferation and lessens chemotherapy-induced myeloma cell line apoptosis (23-25). In a meta-analysis where 1269 MM patients and 2158 controls were evaluated, the circulating LEP levels of MM patients were found to be higher than those of controls (26). Esheba et al. (27) and Alexandrakis et al. (28) also supported these results (29). However, there are also studies showing the opposite (30,31). There are studies relating genetic variants of LEP and LEPR to the susceptibility of various malignant tumors (32). LEP-2548G/A and LEPR 668A/G are the most studied variants. The LEP-2548G/A is close to the specificity protein-1 transcription factor binding site located in the LEP promoter region. This variant is thought to affect LEP gene transcription and expression as well as LEP synthesis and secretion from adipose tissue (12). LEPR 668A/G is located in the leptin-binding region in the extracellular domain of the gene (12). This variant, which disrupts LEP binding to its receptor, results in an A to G substitution in exon 6, nucleotide 668, and codon 223 of the start codon (33). Bieńkiewicz et al. (34) reported that the LEP-2548A/G A/G genotype may reduce the risk of endometrial cancer formation, whereas the A allele may be a risk factor. The LEP-2548A/G polymorphism was linked to an increased overall risk of cancer, according to a meta-analysis (35). A meta-analysis suggested that LEPR 668G/A may play a role in the development of breast cancer in East Asians (36). In addition, it was reported that the LEPR 668G/A G/G genotype was related to an increased LEP profile among obese breast cancer females (33). Another study found that LEPR 668G/A was not associated with cancer in a study of 35,936 subjects in 44 case-control studies (37). There are not many studies examining these variants in hematological malignancies. These two variants are associated with non-Hodgkin lymphoma (38). Lin et al. (39) showed the same result for LEP-2548G/A (39).
In this study, we aimed to investigate whether the LEP-2548A and LEPR-668G/A variants predispose to the development of MM. DNA methylation pattern is an epigenetic change that affects gene expression. Therefore, we also examined the LEP-31 and -51 nt methylation status of the LEP gene. We found that LEP-2548G/A and LEPR 668A/G genotype distributions were not associated with MM development. We found that -31 and -51 nt regions of the LEP gene were unmethylated in the patient group compared with the control group. In multivariate analysis, we found that the methylated/unmethylated ratio at -31 nt methylation was related to a poor prognosis.
Study Limitations
This study has several limitations. First, the sample group was small. Additionally, only two variants in this pathway were evaluated. Other variants in these genes may also play a role in the development of MM. However, it is also an advantage of the study that LEP-2548G/A and LEPR 668A/G variants and the methylation status of -31 and -51 nt in the LEP gene are examined for the first time.
Conclusion
To the best of our knowledge, this is the first study to assess these variations and the methylation status of this region in Turkish individuals with MM. Although the current study’s findings suggest that LEP and LEPR polymorphisms could not be a risk factor for the onset of MM, further research is necessary to validate and expand on these findings in larger populations and other ethnic groups.
Acknowledgments: We respectfully remember all the colleagues we lost in the fight against COVID-19.
Ethics Committee Approval: The study was approved by the İstanbul University, İstanbul Faculty of Medicine Local Ethical Committee (approval number: 62734, date: 29/05/2020).
Informed Consent: It wasn’t obtained.
Authorship Contributions: Concept - Y.O., A.F.N., İ.S., S.P., M.P.; Data Collection or Processing - Y.O., A.F.N., İ.S., S.P., M.P.; Analysis or Interpretation - Y.O., A.F.N., İ.S., S.P., M.P.; Literature Search - Y.O., A.F.N., İ.S., S.P., M.P.; Writing - Y.O., A.F.N., İ.S., S.P., M.P.
Conflict of Interest: No conflict of interest was declared by the authors.
Financial Disclosure: The authors declared that this study received no financial support.