Simple Markers for Subclinical Inflammation in Patients with Familial Mediterranean Fever

Background

Familial Mediterranean fever (FMF) is an autosomal recessively inherited disease characterized by recurrent self-limited attacks of fever accompanied by aseptic inflammation of serosal spaces, joints, and skin. Although acute attacks of this disease are self-limited, some patients develop AA type amyloidosis, leading to renal failure, which is responsible for severe morbidity in FMF patients [1,2].

In FMF, an abnormal form of pyrin, which is a protein encoded by the MEFV gene, causes inflammation. These inflammatory episodes are then mediated by a massive influx of neutrophils into the serous cavities and are accompanied by an elevation in the levels of acute-phase proteins and cytokines [3]. The levels of many blood cytokines and acute phase reactants were studied in FMF patients and contributed to our understanding of the pathogenesis of FMF [4,5]. Further studies demonstrated an activation of the cytokine network in the disease course of FMF. Increased levels of interleukin-6 (IL-6), interleukin-10 (IL-10), serum-soluble interleukin-2 receptor (sIL-2r), and tumor necrosis factor alpha (TNF-α) have been detected during or between acute attacks of FMF in different studies [6,7]. These cytokines are primarily expressed by macrophages and monocytes at inflammatory sites [8]. Recent studies have shown that subclinical inflammation may continue in FMF, even during symptom-free periods [9]. The erythrocyte sedimentation rate (ESR) and acute-phase proteins such as C-reactive protein (CRP), serum amyloid A (SAA), and fibrinogen increase during attack periods and usually return to normal during symptom-free periods [10]. However, amyloidosis can develop in patients during a subclinical period of FMF. NLR, PLR, MPV, and RDW may be indicators of systemic subclinical inflammation [11–13]. NLR, PLR, RDW, and MPV have been associated with conditions such as chronic inflammation in cardiovascular diseases, malignancies, ulcerative colitis, hepatic cirrhosis, and systemic lupus erythematosus. Recent studies have suggested that NLR and MPV are significantly higher in FMF patients [11–15]. The current study was also planned to determine if this association exists between RDW levels and FMF, and to determine which marker – NLR, PLR, MPV, or RDW – best indicates subclinical inflammation in FMF patients. We also aimed to determine if subclinical inflammation markers could act as predictors of amyloidosis.

Material and Methods

This study was conducted retrospectively and included 153 pediatric patients diagnosed with FMF from the pediatric clinic at Gaziosmanpaşa University Medical Practice and Research Center between May 2009 and May 2014. The diagnosis of FMF was made according to the Tel-Hashomer criteria: the presence of at least 1 of 4 major criteria, 2 of 5 minor criteria, 1 minor criterion plus 5 of 10 supportive criteria, or 4 of 5 specific supportive criteria [16]. The control group included 90 age- and sex-matched healthy subjects. Both the study group and the control group subjects had no acute infection, pneumonia, diabetes mellitus, systemic hypertension, acute or chronic renal failure, chronic liver disease, chronic obstructive pulmonary disease, obstructive sleep apnea, coronary artery disease, connective tissue disease, inflammatory bowel disease, allergic rhinitis, asthma history, or any inflammatory disease.

We used an automated blood cell counter and the urine protein/creatinine ratio. A complete blood count (CBC) and biochemical analyses were obtained from hospital records at the clinic. Laboratory findings were collected during symptom-free periods. NLR was noted as a simple ratio between the absolute neutrophil and the absolute lymphocyte counts. NLR, PLR, MPV, and RDW were derived from the CBC.

Subjects were screened for the presence of all 12 known FMF mutations. Genomic DNA was extracted from 5-mL amounts of whole blood obtained via standard procedures. FMF mutations were detected using a reverse-hybridization test strip-based assay (FMF StripAssay, ViennaLab Labordiagnostika, Vienna, Austria), allowing detection of the 12 most common MEFV mutations: p.E148Q (c.442G>C) in exon 2; p.P369S (c.1105C>T) in exon 3; p.F479L (c.1437C>G) in exon 5; and p.M680I (c.2040G>C), p.M680I (c.2040G>A), I692del (c.2076>2078del), p.M694V (c.2080A>G), p.M694I (c.2082G>A), p.K695R (c.2084A>G), p.V726A (c.2177T>C), p.A744S (c.2230G>T), and p.R761H (c.2282G>A) in exon 10.

Continuous variables are presented as mean ±SD and categorical variables are expressed as numbers and percentages. To test the significance between 2 means, we used a comparison of continuous variables between groups. The t test was used for continuous variables with normal distribution and the Mann-Whitney U test was used for continuous variables without normal distribution. The chi-square test was used for categorical variables. Pearson correlation analysis was used to assess the relationships. A p value <0.05 was accepted as statistically significant. A region of conversion (ROC) analysis was used to determine the association of NLR with disease. The relationship between disease and variables was determined with multivariate logistic regression. For statistical calculations, SPSS Statistical Software (SPSS for Windows, version 17.0; SPSS Inc. Chicago, IL, USA) was used.

The Ethics Committee for Clinical Research of Gaziosmanpasa University School of Medicine approved this study. Our study was conducted in accordance with the ethical principles described by the Declaration of Helsinki. The study was supported by Gaziosmanpasa University Health Research and Practice Center.

Results

The study group consisted of 78 boys (51.0%) and 75 girls (49.0%), while the control group had 49 boys (54.4%) and 41 girls (45.6%). The mean age was 12.33±4.41 in the FMF study group and 10.96±3.45 in the control group. Mean follow-up time was 47 months and mean disease onset was 8.41 years. All FMF patients were using colchicine 1–3 times a day. The mean disease severity score was 6.46. The primary characteristics of FMF patients in the study were abdominal pain 96.1% (n=148), fever 84.7% (n=128), arthritis 34% (n=52), pleuritis 14.4% (n=34), erysipelas 4.6% (n=7), appendectomy 17.6% (n=27), and family history 52% (n=79). All of the identified mutations are summarized in Table 1. Thirty-four patients (22.2%) were homozygous, 43 (28.1%) were compound heterozygous, 68 (44.4%) were heterozygous, 5 (3.3%) were wild type, and 3 (1.9%) were not analyzed. The most commonly encountered MEFV gene mutation among the patients was heterozygous.

The 2 groups were evaluated in terms of NLR, PLR, MPV, and RDW values. We found that NLR, PLR, MPV, and RDW levels were significantly higher in the FMF study group than the control group. CRP values in symptom-free patients were significantly higher than in the control group (p<0.001), despite the role of CRP as an acute-phase reactant. NLR, PLR, MPV, RDW, and CRP values of FMF patients with proteinuria and without proteinuria were compared. There was no significant difference between these 2 groups. All laboratory data are summarized in Table 2.

Patients using colchicine 1 mg and under were compared with patients using colchicine over 1 mg. It is seen that higher NLR values required higher colchicine dosage for controlling symptoms (p=0.008).

Patients carrying M694V (either heterozygous or homozygous) mutation were compared with patients carrying other mutations. NLR values of patients with M694V were higher than NLR values in patients with other mutations (p=0.017).

The relationship between disease severity score and NLR was investigated in patients. There was no correlation between these 2 parameters (p=0.343).

There was a positive weak correlation between CRP and either NLR, PLR, MPV, or RDW. NLR had the strongest correlation with CRP levels (Table 3).

NLR showed an area under the curve (AUC) of 0.636 (95% CI, 0.569–0.698) with a cut-off value of 1.65 based on ROC analysis. This was found to be a reliable but simple marker for subclinical inflammation, as shown in Figure 1.

Discussion

LIMITATION OF THE STUDY:

It would have been useful to investigate the NLR values in patients during the attack period, but we could not compare patients in attack and attack-free periods because all of our included patients in this study were in the attack-free period. We would have compared the values of patients with amyloidosis, but only 1 of our patients was followed-up with amyloidosis secondary to FMF.

Conclusions

Although NLR, PLR, MPV, and RDW cannot be used to detect early signs of proteinuria, NLR, PLR, MPV, and RDW levels can be used to determine subclinical inflammation. However, we found that NLR had the strongest correlation with subclinical inflammation with CRP among these parameters. In this relationship we found that the cut-off value of NLR is 1.65 as a numerical marker for subclinical inflammation. Subclinical inflammation goes on in attack-free periods and causes amyloidosis. We suggest that NLR may be useful as a predictor of development of amyloidosis.