Rheumatology

Rheumatol Int DOI 10.1007/s00296-014-3116-x

INTERNATIONAL

Original Article - Genes and Disease

Familial Mediterranean fever in Syrian children: phenotype–genotype correlation Rami A. Jarjour · Sumaya Al‑Berrawi 

Received: 28 May 2014 / Accepted: 13 August 2014 © Springer-Verlag Berlin Heidelberg 2014

Abstract  Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent attacks of serosal membranes. In this study, 103 unrelated Syrian children were included. Mutation screening of the MEditerranean FeVer gene was performed for 12 mutations. Abdominal pain was observed in 91 (88.3 %) of the patients, fever in 82 (79.6 %), arthritis in 27 (26.2 %), pleuritis in 7 (6.7.5 %), rash and erysipelas-like erythema in 5 (4.8 %), myalgia in 5 (4.8 %), headache in 5 (4.8 %) and Henoch–Schonlein purpura in 1 (0.97 %). The most frequent mutation was M694V. In order to determine the association between M694V and clinical features of FMF, we compared the disease features between patients with and without this mutation. The presence of M694V was found to be associated with more severe course of FMF, earlier age of onset and more frequent arthritis in the Syrian children with FMF compared to other FMF patients who do not have this mutation. Keywords  Familial Mediterranean fever · Genotype–phenotype correlation · Syrian children

Introduction Familial Mediterranean fever (FMF) (OMIM #249100) is a genetic disease with autosomal recessive inheritance. The disease most commonly occurs in Arabs, Jews, Armenians and Turks [1]. FMF is clinically characterized by recurrent

R. A. Jarjour (*) · S. Al‑Berrawi  Clinical Genetics Unit, Molecular Biology and Biotechnology Department, Atomic Energy Commission of Syria (AECS), P.O.Box 6091, Damascus, Syria e-mail: [email protected]

and self-limited attacks of fever accompanied by peritonitis, pleuritis, synovitis or erysipelas-like erythema and may be complicated by AA amyloidosis. FMF patients are free of symptoms between attacks. However, the clinical picture of FMF has been expanded and additional features have been described including pericarditis, myalgia and scrotal swelling [2]. Colchicine was found to be very effective in preventing attacks of FMF and in the development of amyloidosis [3, 4]. The responsible gene, MEditerranean FeVer (MEFV), has been mapped to chromosome 16p13.3 and consists of 10 exons and encodes a 781 amino acid protein called Pyrin which is expressed in granulocytes and is thought to be a negative regulator of inflammation [5, 6]. Since the cloning of the MEFV gene, about 166 mutations have been associated with FMF [7]. Five founding mutations M694V, V726A, M680I, M694I (in exon 10) and E148Q (in exon 2) are the most frequently encountered mutations and account for 74 % of FMF mutations in typical patients [8]. Phenotype–genotype correlation has not been explained definitely and awaits to be clarified. Several reports have shown an association between mutation M694V and the severe course of FMF with early onset, high frequency of attacks, the need to a high dose of colchicine and high frequent occurrence of amyloidosis in untreated patients [9– 15]. However, this is still a controversial issue as others did not find any association between genotype and phenotype [16, 17]. FMF is common in Syrians, and the carrier rate is 17.5 % [9, 18]. In the Syrian FMF patients, the most frequent mutation was M694V (36.5 %), followed by V726A (15.2 %), E148Q (14.5 %), M680I (G/C) (13.2 %) and M694I (10.2 %) mutations [19]. The main aim of this study was to study the clinical features of FMF in Syrian children and to investigate the phenotype–genotype correlation in

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the same group of patients. To the best of our knowledge, this is the first report from Syria regarding FMF in children.

Patients and methods A total of 103 unrelated children were referred by their pediatricians for FMF mutation detection and genetic counseling. Information was obtained from the parents in the presence of their children. A clinical diagnosis of FMF was made according to the Tel Hashomer criteria [20]. The disease severity was evaluated according to the previously described criteria [21]. Every family was informed about the study and a written consent was signed by one of the parents for blood sampling. For all patients, EDTA blood was sampled and DNA was isolated from frozen blood samples by the phenol– chloroform extraction method [22]. MEFV mutation analysis was performed for 12 mutations using a reverse-hybridization assay (FMF StirpAssay™) according to the manufacturer’s instructions (ViennaLab Labordiagnostika, Vienna, Austria). In a first step, exons 2, 3, 5 and 10 were amplified for each patient in a single, multiplex PCR reaction. A thermocycling program of 35 cycles (94 °C for 15 s, 58 °C for 30 s and 72 °C for 30 s) with a final extension at 72 °C for 3 min was performed, leading to four DNA fragments (206, 236, 295 and 318 bp). PCR products were selectively hybridized to a test strip presenting a parallel array of allele-specific oligonucleotide probes and detected by enzymatic color reaction as described by Tchernitchko et al. [23]. The following MEFV mutations were investigated: E148Q in exon 2, P369S in exon 3, F479L in exon 5, and M680I (G/C), M680I (G/A), I692del, M694V, M694I, K695R, V726A, A744S, R761H in exon 10. Statistical analysis Differences between groups were assessed using the Z test for two proportions and T test for means. A P value 10 Total

No.

%

60 28 15

58.3 27.2 14.5

103

100

whereas the parents of 41 (39.8 %) patients proved to be relatives. The main clinical characteristics of the patients were as follows: Peritonitis was observed in 91 (88.3 %) of the patients, fever in 82 (79.6 %), arthritis in 27 (26.2 %), pleuritis in 7 (6.7.5 %), rash and erysipelas-like erythema in 5 (4.8 %), myalgia in 5 (4.8 %), headache in 5 (4.8 %), purpura in 1 (0.97 %) and Henoch–Schonlein purpura in 1 (0.97 %). No patient had pericarditis or scrotal swelling. None of the patients developed amyloidosis. Among the 103 patients, 32 (31 %) were treated with colchicine and 24 (75 %) responded with complete remission or significant improvement (reduction of number and severity of FMF episodes). Mutations in the MEFV gene were identified in 74 (71.8 %) patients, and the mutation detection was negative in 29 (28.2 %) patients. Of those 74 patients with mutations, 14 (18.9 %) were homozygous, 27 (36.5 %) were compound heterozygous, and 33 (44.6 %) were heterozygous (Table 2). Complex alleles (>2 alleles) were observed in five patients (E148Q/M680I(G/A)/M694I, E148Q/F479L/V726A, E148Q/M680I(G/C)/M694V/ M694I, M694V/I692del/E148Q and E148Q/M680I(G/C)/ M680I(G/C) (Table 2). Mutation detection allowed the identification of 12 different MEFV mutations in 121 of the 148 tested alleles (Table 3). The most dominant mutations detected were M694V (36.4 %), E148Q (14.9 %), M680I (G/C) (14 %), M694I (11.6 %) and V726A (10.7 %) (Table 3). The remaining seven mutations (R761H, A744S, M680I (G/A), K695R, P369S, F479L and I692del) were much less represented (Table 3). In order to evaluate the phenotype–genotype correlation, 74 patients were divided into three groups according to the presence of the M694V mutation on both of the alleles (M694V/M694V), on only one allele (M694V/ other), and on none of the alleles (other/other). Among those patients, 12 clinical features of the disease were compared (Table 4). There were no statistically significant differences between these groups with regard to male/ female ratio, abdominal pain, fever, chest pain, erysipelaslike erythema, myalgia, headache, purpura and Henoch– Schonlein purpura and family history of FMF (Table 4). However, there were statistically significant differences between groups I and III, and groups II and III regarding

Rheumatol Int Table 2  Genotypes of Syrian children with FMF: homozygotes, compound heterozygotes and heterozygotes

Table 3  The distribution of the 12 MEFV mutations screened in 74 Syrian children

No.

Number of mutations in FMF patients N (%)

Homozygotes  1  2  3  Subtotal Compound heterozygotes  4  5  6  7  8  9  10  11  12  13  14  15  16  17  18  19  20  Subtotal Heterozygotes  21  22  23  24  25  26  27  28  29 Subtotal Total patients with mutations Patients with no identified mutations Total number of patients

Genotype

M694V/M694V M680I(G/C)/M680I(G/C) M694I/M694I

Patients n (%)

11 2 1 14

M694V/V726A M694V/M680I(G/C) M694V/R761H M694V/E148Q M694V/A744S M694V/M694I M680I(G/C)/R761H E148Q/R761H M680I(G/C)/V726A M680I(G/C)/E148Q M694I/V726A M694I/M680I(G/C)

6 2 3 2 1 1 1 1 1 1 1 2

E148Q/M680I(G/A)/M694I E148Q/F479L/V726A E148Q/M680I(G/C)/M694V/ M694I M694V/I692del/E148Q E148Q/M680I(G/C)/M680I(G/C)

1 1 1

E148Q M694V M694I V726A M680I(G/C) K695R P369S A744S R761H

1 1 27 9 5 6 4 3 2 1 2 1 33 74 29 103

age of onset and arthritis. The disease severity score was calculated according to the Tel Hashomer criteria [21]. There were statistically significant differences between the mean severity scores of groups I and III, and groups II and III (Table 4).

M694V E148Q M680I(G/C) M694I V726A R761H A744S K695R M680I(G/A) P369S F479L I692del Total

Mutation 44 (36.4 %) 18 (14.9 %) 17 (14 %) 14 (11.6 %) 13 (10.7 %) 6 (5 %) 3 (2.5 %) 2 (1.7 %) 1 (0.8 %) 1 (0.8 %) 1 (0.8 %) 1 (0.8 %) 121 (100 %)

Discussion In this study, we evaluated the clinical features and the genotypes of FMF in Syrian children. The frequency of FMF was similar in both genders (female to male ratio was 1.01). The prevalence of FMF in Arabs is roughly equal in men and women, whereas in other ethnic groups the prevalence is higher in men [24]. Our data do not support the suggestion that FMF may have incomplete penetrance in female patients [25]. For 60 (58.3 %) children, FMF had started before the age of 5 years, and in 88 (85.5 %) FMF started below the age of 10 years. About 80 % of Arab patients with FMF present before age 10 years [26, 27]. Abdominal pain is the most common feature occurring in about 82–96 % of FMF patients [28]. In our patients, abdominal pain was observed in 88.3 % of the patients. Fever is also a common feature of acute FMF episodes (the frequency ranges from 93 to 100 %) [28]. Of our patients, 79.6 % had fever. Arthritis is a common and important feature of FMF. It was detected in 26.2 % of the patients. Three patients had arthritis as the only clinical feature of FMF. Sometimes arthritis remains the only manifestation of FMF [17]. None of the patients had protracted arthritis. The frequency of arthritis varies between different ethnic groups. Arthritis occurs in 77 % of Sephardic Jews [1], whereas the incidence is significantly lower in Arabs (37 %) [29], Turks (47 %) [30] and Armenians (37 %) [31]. No patient had pericarditis or scrotal swelling. Pericarditis was reported in 0.5 % of FMF patients [32]. Erysipelaslike erythema is the most characteristic coetaneous lesion in FMF [33]. It occurs below the knees on the anterior leg or dorsum of the foot (unilateral or bilateral). Frequency

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Rheumatol Int

Table 4  Clinical features of subgroups according to the M694V mutation M694V/M694V Group I (No. = 11)

M694V/other Group II (No. = 22)

Other/other Group III (No. = 41)

Significance Groups I and II

Groups I and III

Groups II and III

NS

NS NS NS NS NS NS NS

1. Male/female 2. Age of onset

4/7 3.35 ± 1.91

9/13 3.88 ± 2.5

19/22 6.16 ± 3.790

NS Sb

3. Abdominal pain 4. Fever 5. Chest pain 6. Arthritis 7. Erysipelas-like erythema 8. Myalgia 9. Headache 10. Purpura and Henoch–Schonlein purpura 11. Family history of FMF

8 (72.7 %) 10 (90 %) – 7 (63.64 %) –

17 (72.27 %) 18 (82.6 %) 2 (9.09 %) 11 (50 %) 2 (9.09 %)

38 (92.6 %) 29 (70.7 %) 3 (7.3 %) 7 (17.1 %) 2 (4.8 %)

NS NS NS Sd NS

Sa NS NS NS Sc NS

2 (18.18 %) 1 (9.09 %) –

1 (4.55 %) 1 (4.55 %) 2 (9.09 %)

1 (2.4 %) 1 (2.4 %) –

NS NS NS

NS NS NS

NS NS NS

3 (27.27 %)

8 (36.36 %)

12 (29.2 %)

NS

NS

NS

12. Mean severity score 7.36 ± 1.56

6.45 ± 1.26

5.24 ± 1.3

Sf

Se

NS

a

  P value 0.0129; b P value 0.0097; c Z value 2.709; d Z value 2.466; e P value