Introduction

Familial Mediterranean fever (FMF) is an inherited autoinflammatory disease characterized by recurrent self-limited attacks of fever and serositis accompanied occasionally by rashes and arthritis [1]. FMF is caused by mutations in the MEFV gene, which is located on chromosome 16p13.3. The MEFV gene comprises 10 exons and encodes pyrin, a protein involved in the regulation of apoptosis and inflammation. Over 80 mutations have been identified so far. Four missense mutations (M680I, M694V, M694I, and V726A) in exon 10, together with E148Q in exon 2, account for the majority of FMF mutations in populations originating from areas around the eastern Mediterranean region [2]. The various combinations of MEFV mutations are largely associated with the phenotypic variability of the disease. The most serious complication of FMF is the development of renal amyloidosis, which may be the only manifestation of the disease [35].

A connection between MEFV mutations and vasculitis such as Henoch–Schönlein purpura (HSP), polyarteritis nodosa, and Behçet disease has been increasingly reported in recent years [68].

HSP is the most common systemic small-vessel vasculitis in childhood, involving the skin, gastrointestinal (GI) tract, joints, and kidney [9]. It was reported that HSP occurs in 7 % of Turkish patients with FMF. The patients with HSP may develop symptoms related to FMF years after the presentation of HSP [6]. Because FMF is common in the Mediterranean region, we investigated MEFV mutations in patients presenting with HSP without any typical FMF symptoms and whether these mutations have any effect on the disease course or complications.

Materials and methods

Clinical and laboratory data of 76 pediatric patients meeting the criteria of the international study group for the diagnosis of HSP [10], who were followed at our institution between September 2000 and October 2010, were analyzed retrospectively. Two patients with established diagnosis of FMF before the diagnosis of HSP were excluded from the study. No patient had a family history or suspected diagnosis of FMF. For the diagnosis of FMF, Livneh criteria were used [1].

In all patients, the symptoms and signs of skin, GI tract, joint, and renal involvement were recorded on admission or at follow-up. Cutaneous findings consisted of characteristic palpable purpura and subcutaneous edema. GI manifestations included bowel angina and GI bleeding defined as the presence of melena, hematochezia, or occult blood in the stool. All patients were followed for a minimum period of 6 months for renal involvement, which was classified as microscopic hematuria, mild proteinuria (<20 mg/m2/h) and/or hematuria, nephrotic range proteinuria, nephrotic syndrome, nephritic syndrome, and nephritic–nephrotic (mixed) syndrome according to the initial clinical presentation. White blood cell (WBC) count, C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), stool blood analysis, and other laboratory parameters were determined by standard laboratory methods at the time of diagnosis. Blood samples for mutation analysis and informed consent from the parents of the children were obtained during follow-up visits. The patients were divided into two groups according to the presence of MEFV mutations as Group 1: patients without a mutation and group 2: MEFV mutation carriers (patients with mutations in at least one allele; heterozygous, homozygous, and compound heterozygous).

The study was approved by the local Ethics Committee.

Detection of the MEFV gene mutations

Each sample was screened for the mutations located in exon 2 and exon 10 of the MEFV gene by direct sequencing. Genomic DNA was extracted from ethylenediamine–tetraacetic acid—anticoagulated whole blood samples using Genomic DNA Purification Kit (Invitrogen, Carlsbad, CA, USA). Following selective amplification of exon 2 and exon 10 with specific primers (primer sequences available upon request), polymerase chain reaction (PCR) products were run in a 2 % agarose gel containing ethidium bromide and visualized under UV light by an imaging system (SynGene InGenius, Cambridge, UK). Then, sequencing was performed using the BigDye Terminator v3.1 Cycle Sequencing Kit and an ABI prism 3130 Genetic Analyzer (Applied-Biosystems, Foster City, CA, USA).

Statistical analysis

All statistical analyses were performed using Statistical Package for the Social Science for Windows 16.0 (SPSS, version 16). All data are expressed as median, mean ± standard deviation, or percentage. Mann–Whitney, Student’s t test, Fisher’s exact test, and Yates’ continuity correction test were used, and values of p < 0.05 were accepted as statistically significant.

Results

The patient population consisted of 76 children (46 boys, 30 girls). Mean age at the time of diagnosis was 7.9 ± 3.7 years (range, 1.5–17.9 years). Palpable purpura, arthritis, GI involvement, edema, and renal involvement were found in 100, 78.9, 35.5, 31.5, and 28.9 % of the patients, respectively. The clinical data of the patients are shown in Table 1.

Table 1 Clinical data of 76 patients

Of 76 patients, 58 (76.3 %) had no MEFV mutations, 11 (14.4 %) were heterozygous, and 7 (9.2 %) had mutations in both alleles, 5 of whom were homozygous and the remaining 2 were compound heterozygous (Table 2). Altogether, 25 mutated alleles were found in 18 patients, showing that the mutated allele frequency is 0.16.

Table 2 Distribution of the MEFV gene mutations

Mean age at diagnosis and sex distribution of the groups were similar. Joint, renal, GI involvement, subcutaneous edema, as well as leukocyte counts, ESR or serum CRP concentrations were also similar among the groups (Table 3). Mean follow-up time of four patients with renal involvement in group 2 was 2.6 ± 1.1 years (range: 1.4–4.2 years), all of whom were in complete remission at the time of study. Clinical and laboratory features of the patients with renal involvement are shown on Table 4. Two of 4 patients with M694 V homozygous mutation had arthritis, one had bowel angina, and one had nephrotic range proteinuria during vasculitis episode. Because the patient who developed nephrotic range proteinuria did not respond to steroid treatment, cyclosporine A was used for 2 years. Her treatment had finished at the time of study, and her renal functions were preserved without proteinuria. No patient developed FMF during the follow-up period.

Table 3 Clinical and laboratory features and MEFV mutations of 76 patients
Table 4 Clinical and laboratory features of the patients with renal involvement

Discussion

Recent publications have reported that the prevalence of MEFV gene mutations in children presenting with HSP was higher than that of the general population [1113] and that MEFV gene mutations could affect clinical and laboratory findings of HSP [12, 13].

It was reported in a previous study that the carrier frequency of five common MEFV gene mutations in the general Turkish population was 20 % (3 % for M694V, 5 % for M680I, 2 % for V726A, 0 % for M694I, and 12 % for E148Q mutation) and that the mutated allele frequency was 0.11 [14]. In our study, mutated allele frequency was found to be 0.16. Although this ratio was higher than that of the general Turkish population, the association was not statistically significant (p = 0.11, Yates’ continuity correction test). However, we found that the frequency of homozygous and compound heterozygous MEFV mutations (9.2 %) was higher than that of the general Turkish population (1 %) (p = 0.026, Yates’ continuity correction test). Of these mutations, four were M694V/M694V, associated with a severe phenotype and amyloidosis, whereas two were V726A/V726A and E148Q/M694V, associated with a mild form of the disease, and the remaining one was L110P/E148Q, which is very rare in the Turkish population.

In a study from Turkey, Ozcakar et al. [12] found that 34 % of 80 patients with HSP had heterozygous MEFV mutations, whereas none of the patients had homozygous or compound heterozygous mutations. In another study from Turkey, Bayram et al. [13] demonstrated that heterozygous and homozygous/compound heterozygous mutation rates were 31 and 13 % in 107 patients, respectively. In a study from Israel, Gershoni-Baruch et al. [11] found that 27 % of 52 patients carried at least one mutation. In all three studies, the prevalence of MEFV mutations of patients with HSP was significantly higher than that of the general population, contrary to our study [1113].

In the present study, subcutaneous edema existed in 50 % of mutation-positive patients. When mutation-positive and mutation-negative patients were compared, edema was more frequent in mutation-positive patients, although statistically insignificant (p = 0.054). Ozcakar et al. [12] found that edema was statistically more frequent in patients with heterozygous mutations in their study group. Bayram et al. [13] found that urogenital involvement was higher in mutation-positive patients; however, they found that the frequency of edema was not different between groups. The results from Israel [11] showed that there were no statistically significant differences in clinical manifestations between groups with and without mutations.

Overall prognosis of HSP is relatively good, and the long-term prognosis depends on renal involvement. The incidence of HSP nephritis (HSPN) has been reported to be 15–62 %, and end-stage renal disease occurs 1–7 % of HSPN patients [15, 16]. Having nephritic/nephrotic syndrome and/or more than 50 % of crescentic or sclerosing glomeruli are considered as the risk factors of long-term renal impairment [17, 18]. In our study, we did not find statistically significant correlation between MEFV mutation carriage and renal involvement. The lack of association may be explained by the small number of the patients.

In two studies from our country, it was reported that acute phase reactants were higher in the mutation carrier group [12, 13]. In our study, acute phase reactants including leukocyte counts, ESR, and serum CRP concentrations were not statistically different between the groups.

In conclusion, it seems that MEFV gene mutations may not affect the long-term prognosis and clinical presentation of HSP. However, further prospective studies are needed to confirm our results.