INTRODUCTION

Familial Mediterranean fever (FMF) is a recessively inherited autosomal inflammatory disease. It particularly affects people in the Mediterranean region, such as Turks, Armenians, Arabs, and non-Ashkenazi Jews. Its characteristics include recurrent episodes of fever, peritonitis, arthritis, rashes, and other serosal inflammations (1). This disease is caused by MEFV mutations, located on the chromosome 16 short arm (2). V726A, M694I, E148Q, M680I, and M694V, mostly located on exon 10, are the mutations of the MEFV gene with the highest frequency (3). Mutation of the pyrin protein encoded by the MEFV gene has been suggested to cause uncontrolled inflammation.

Nitric oxide (NO), as a gaseous free radical, is synthesized from oxygen and l-arginine and by four main isoforms of NO synthase (NOS), including endothelial NOS (eNOS), neuronal NOS, inducible NOS, and more recently, mitochondrial NOS (4). NO powerfully regulates the immune response, activating a cascade of signal transduction pathways involved in autoimmune and inflammatory responses (5). The eNOS gene is located on chromosome 7q35-36. It consists of 26 exons spanning a 21 kb genomic region. Its primary expression is in the low-level endothelial cells in platelets (6). The eNOS can stimulate cyclooxygenase 2, KB nuclear factor, and pro-inflammatory cytokines (7). The plasma NO level in healthy people is linked to the variations in the eNOS gene. There are different polymorphic sites in the eNOS, one of which is VNTR (27 bp repeat, intron 4 VNTR a/b), which causes it to produce basal NO. Due to the possible bonding of the endothelial nuclear proteins to this region, the eNOS gene promoter efficiency and then the levels of eNOS protein and enzyme activity may be influenced by the VNTR (8). There has been a report of the VNTR five alleles, with 2-6 tandem 27-bp repeats (alleles 2-6) published up to date, among which are alleles 4 and 5 in all populations under study (9). It is shown that the concentration and activity of eNOS are lower in the heterozygotes of the 4a/4b variant than those in the homozygotes of 4b/4b (9).

Ongoing chronic inflammation causes endothelial dysfunction. There is a relationship between endothelial dysfunction and the defects in endothelium-based vasodilation mediated by NO (10). It has been reported that endothelial biomarkers are abnormal in patients with FMF.

As the frequency of variants varies between ethnic groups and races, we examined the genotypic and allelic distribution of the eNOS VNTR variant in FMF patients in this study. We also evaluated the association between the eNOS VNTR variant and MEFV gene mutations.

METHODS

Study Population

The study population included 160 unrelated patients with FMF (89 females and 71 males) who attended the Department of Medical Genetics, Samsun Research and Training Hospital, University of Health Sciences Türkiye, Samsun, Türkiye. Five mutations were examined in patients (M694V, M680I, V726A, E148Q, and A744S). FMF was diagnosed on the basis of the Tel Hashomer criteria. A group of 147 healthy Turkish volunteers (80 females and 67 males) matched by age, sex, and ethnicity with no history or signs of FMF or inflammatory diseases were included as the control group. All the subjects lived in the Central Black Sea region and were over 18 years old. Each patient had detailed clinical characteristics that were recorded. All participants were informed about the study and gave their written informed consent for a protocol. This study was conducted on the basis of the Declaration of Helsinki. The study protocol was approved by the University of Health Sciences Türkiye, Samsun Training and Research Hospital Clinical Research Ethics Committee (protocol no: KAEK 2020/5/13, date: 16.06.2020).

Genotyping

As instructed by the manufacturer, 2-mL blood was taken from each participant, including the controls and the FMF patients, and the commercial kit was used to extract DNA from all samples. eNOS VNTR variant was genotyped using the polymerase chain reaction method, which was described previously method (11).

String Analysis

The functional interactions among the proteins are annotated by the STRING database in a cell. In this study, we analyzed the VEGF protein with the STRING database.

Statistical Analysis

The Statistical Package for Social Sciences (SPSS) software version 20.0 for Windows (SPSS, Inc., Chicago, IL) was used for the analysis of the data. The results are presented as the mean standard deviation (SD). Fisher’s Exact test, the χ² test, or ANOVA were used to analyze the relationships between the eNOS VNTR variant and the patients’ demographic and clinical characteristics. Risk factors were assessed using 95% confidence intervals (CIs) and odds ratios (ORs). There were two-tailed p-values, and p-values below 0.05 were regarded as significant.

RESULTS

In this study, 307 subjects, including 147 adult controls and 160 FMF patients, were genotyped for the eNOS VNTR variant. The mean age ± SD was 27.23±10.90 in the patients and 37.39±13.39 in the control group. Eighty nine (55.63%) women and 71 (44.37%) men were included in the patient group, and 80 (54.42%) women and 67 (45.58%) men were in the control group. Table 1 shows the patient and control groups’ demographic and baseline clinical features.

We evaluated MEFV mutation distributions in FMF patients. 51.9% of patients had two or more MEFV mutations. The most prevalent mutation was the homozygous M694V/M694V mutation (25%). The distribution of MEFV mutations in patients is presented in Table 2.

The genotype and allele frequencies of the eNOS gene VNTR variant in FMF patients and healthy controls are given in Table 3. A significant difference was found in the distribution of genotypes of the eNOS VNTR variant between patients and controls. eNOS VNTR 4a/4a and 4b/4b genotypes were higher in patients compared to controls, respectively [p (p˂0.05, OR 2.755, 95% CI: 1.380-5.021; p˂0.05, OR 1.167, 95% CI: 0.928-1.452)]. The patients and the controls did not show significantly different allele frequencies of the eNOS VNTR variant (p>0.05). 

We then evaluated the relationship between the distribution of the MEFV mutations and the genotype distribution of eNOS VNTR in the FMF patients (Table 4). No significant difference was found between MEFV mutations and eNOS VNTR variants in the patients (p>0.05).

We then evaluated the association between the clinical features of FMF and the eNOS VNTR genotype distribution (Table 5). Patients with the 4b/4b genotype had higher colchicine usage and responses (p>0.05).

When we analyzed the eNOS protein with the STRING database, we predicted the protein’s functional partners with high confidence (score: 0.7) as follows: ESR1, KDR, VGFA, HSP90AA1, AKT1, CALM1, CAV1, KNG1, NOSTRIN. The interaction network of these proteins is shown in Figure 1.

DISCUSSION

FMF has been classified as a systemic autoinflammatory disorder (12). This disease is common in the Turkish population. Despite the discovery of the responsible gene (MEFV), there is no clear link between genotype and phenotype. The MEFV gene has a predominant expression in neutrophils (13). Pyrin encoded by MEFV forms the NLRP3 inflammasome complex element and modulates the pro-inflammatory cytokine interleukin-1 (IL-1b) generation. Therefore, FMF may be regarded as inflammasompathy (14). 

Initially, IL-1b is released as a proinflammatory cytokine during attacks, significantly increasing the serum levels of acute-phase reactants such as C-reactive protein (CRP), fibrinogen, erythrocyte sedimentation rates (ESRs), and serum amyloid A (15). The endothelial adhesion molecules, including intercellular adhesion molecule-1 and vascular cell adhesion molecule-1, can facilitate the cellular interactions between B- and T-lymphocytes, monocytes/macrophages, and neutrophils. Endothelial cells can be facilitated by a rich source of proteolytic enzymes and oxygen reactive species, i.e., neutrophils, which are against junctions of the end. This endothelial dysfunction basic mechanism, which neutrophils initiated causes various inflammatory disorders, including FMF (16,17). The pathogenesis of the disease may be caused by several environmental factors and other modifying genes. Studies have found an association between different inflammatory gene variants and FMF (18,19).

Host defense and homeostasis are affected by NO when generated at a low level in the short term; but when it is generated at higher concentrations in the longer term, it becomes mutagenic and genotoxic. For this reason, the NO-mediated effects have complex biological outcomes depending on the external and internal environment of the cell’s generation and target sites, as well as the generated NO concentration (20). Different inflammatory diseases such as rheumatoid arthritis (21), osteoarthritis (22), Sjogren’s syndrome (23), and systemic lupus erythematosus (SLE) (24) show excessive NO production. Balat et al. (25) reported that total nitrite levels were significantly higher in children with FMF. The endothelial NO production is primarily affected by eNOS activity. eNOS, which is an enzyme dependent on Ca2+, was first defined in the vascular endothelial cells. eNOS function and activity largely affect endothelial function (26). Endothelial dysfunction is heavily affected by oxidative stress caused by eNOS dysfunction.

TNF-a, IL-17, cluster of differentiation 40 ligand, interferons, CRP, and SLE-specific circulatory factors have promoted endothelial dysfunction by promoting abnormal eNOS function and increasing oxidative stress in recent studies (27). Oxidative stress increased in patients with FMF during attack (28). Yel et al. (29) reported the presence of endothelial damage, particularly during the active disease period, among children with FMF. Additionally, there is little information on endothelial dysfunction and renal involvement in FMF (10). There are two common alleles in the eNOS gene 4a/b variant of the 27-bp VNTR in intron 4: 4b with 5 repeats and 4a with 4 repeats (30). The eNOS VNTR variant has been suggested to regulate eNOS expression by forming small RNAs (siRNAs). There are lower levels of eNOS and higher quantities of siRNA in the endothelial cells containing five copies than in the cells containing four copies (31). The eNOS VNTR variant has been linked to many inflammatory diseases, including autoimmunity diseases such as SLE (32) and Behçet’s disease (33).

In this study, we evaluated the distribution of the eNOS VNTR variant among Turkish FMF patients and whether this variant is a risk factor for developing FMF. As far as we know, this is the first study on the prevalence of the eNOS VNTR variant among Turkish patients with FMF. There was a statistical difference between FMF patients and controls in terms of eNOS VNTR variant genotype distribution (Table 2). eNOS VNTR homozygous genotypes (4a/4a and 4b/4b) were more common in patients than in controls. When we compared clinical findings with eNOS VNTR genotypes, we found that eNOS VNTR genotype distribution was associated with the response to colchicine and colchicine usage (Table 5). The responses to colchicine and colchicine usage were higher in patients with the 4b/4b genotype.

Our study had some limitations. First, we could not evaluate other acute complications of FMF. The other one was that our sample size was small.

CONCLUSION

NO’s the ability to regulate immune responses has been remarkable over the last two decades. Every cell type in the body virtually produces NO, which is effective in regulating processes at a larger scale, such as the immune and nervous systems. The central issue in FMF is the dysfunction of the innate immune system as a self-reactive autoinflammatory disease. Despite intense research on the pathogenesis of FMF, there are still unknowns. Our results show that the eNOS VNTR variant is a risk factor for FMF susceptibility.

ETHICS

Ethics Committee Approval: The study protocol was approved by the University of Health Sciences Türkiye, Samsun Training and Research Hospital Clinical Research Ethics Committee (protocol no: KAEK 2020/5/13, date: 16.06.2020).

Informed Consent: All participants were informed about the study and gave their written informed consent for a protocol.

Authorship Contributions

Concept: Ö.S.T., S.Y., Design: A.F.N., A.T., Data Collection or Processing: Ö.S.T., Analysis or Interpretation: S.Y., A.T., Literature Search: A.F.N., Writing: A.F.N.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.