Familial Mediterranean Fever (FMF) is the commonest monogenic auto-inflammatory disease. The clinical features include recurrent self-limiting attacks of fever, peritonitis, pleuritis, arthritis, and cutaneous rashes. Amyloidosis is the most significant complication of FMF, leading to kidney failure (1). The first attack and symptoms typically occur in early childhood. FMF is mainly observed in individuals of Mediterranean descent, including Turks, Greeks, Jews, Arabs, Armenians, and Persians. The prevalence of FMF in endemic countries is about 1:500 to 1:1000, and it is rare in other ethnic populations (2). The precise prevalence of FMF in Iran is yet to be determined, highlighting the need for further research, but the estimated carrier rate is 27.4 % (3).
Serum amyloid-A protein (SAA), haptoglobin, ceruloplasmin, erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), fibrinogen, leukocyte count, and some cytokines are acute phase reactants that rise in the blood during attack periods. These findings may help support the diagnosis or rule out other causes (4).
The diagnosis of FMF, especially in endemic regions, is generally clinical, using the Tel Hashomer criteria (5). Genetic evaluations are crucial for confirming the diagnosis in patients with the atypical phenotype of FMF. However, 10% to 20% of patients showing typical clinical manifestations lack a mutation in the MEFV gene (6).
Mutations in the Mediterranean fever gene (MEFV) cause FMF. MEFV is located on chromosome 16p13.3, consists of 10 exons, and encodes the protein pyrin/marnosterin. Pyrin is expressed mainly in monocytes, neutrophils, eosinophils, and fibroblasts, which play an essential role in inflammatory responses by regulating caspase-1 activation and processing of mature IL-1β (2).
Mutations in the MEFV gene cause abnormal synthesis of pyrin proteins. In mutated conditions, inflammatory regulation does not occur correctly. Colchicine is the therapy of choice for patients with FMF as it inhibits attacks and the progression of amyloidosis. Interleukin-1 (IL-1) inhibitors are the first-line alternatives in colchicine non-responder/intolerant FMF patients (7).
So far, the Infevers database (8) has recorded 399 mutations and polymorphisms in the MEFV gene, mostly located in exons 2, 10, 5, 3, and 1, with over 96% being substitutions. The distribution of MEFV gene variants differs across different ethnic groups. In Iranian patients with FMF, the most prevalent mutations are p.M694V, p.E148Q, p.V726A, p.R761H, p.M680I (both c.2040G>C and c.2040G>A), and p.M694I (9).
So far, several studies from different regions of Iran have examined the spectrum of MEFV gene variants in patients with FMF. Limited studies have been done in southwest Iran, underscoring the need for additional research in this region. In the present study, we sequenced all 10 exons of the MEFV gene in 40 unrelated pediatric patients with clinical suspicion of FMF in the Southwest of Iran. We aimed to explore all mutations in all exons of the MEFV gene and compare them with the spectrum of MEFV gene variants in other regions of Iran and other countries, considering the patients' clinical phenotypes.

  

Discussion

To confirm the diagnosis of FMF disease, molecular investigation of the MEFV gene and identification of relevant mutations are necessary. This study confirmed FMF in 16 out of 40 unrelated pediatric patients. We identified six common mutations, including p.E148Q, p.M694V, p.M694I, p.A744S, p.V726A, and p.M680I (c.2082G>A). We also identified one uncommon mutation (p.R202Q) and one rare mutation (p.S339F) in our patients. Although several reports from Iran described p.M694V as the most common mutation (9), p.E148Q was the most prevalent mutation in our patients (50%). Similar to our research, the studies of Haghighat et al., conducted on southwest Iranian patients in 2017 (n=20), and Malek et al., performed on northeast Iranian patients in 2023 (n=29), found that the most frequent mutation was p.E148Q, with a frequency of 25% and 41.3%, respectively (11, 12).
This study detected the rare mutation p.S339F, representing the first case reported in Iranian populations. Our study identified this rare mutation in P9 as a compound heterozygote with p.E148Q (a 13-year-old boy). When he was six years old, he had recurrent episodes of abdominal pain, fever, and myalgia. The patient responded to treatment with colchicine. This mutation was registered for the first time in 2006 from Turkey on the Infever database (8). Thus far, it has been reported in only a few patients worldwide. In the two separate studies, one including 15 patients by Stella et al. in 2019 from Italy and the other including 56 subjects by Cetin et al. in 2020 from Turkey, the mutation p.S339F was reported in combination with one or two other mutations in the MEFV gene (p.A744S/p.S339F/p.Asn256Argfs*70 and p.S339F/p.R202Q). However, the detailed clinical features of the reported patients were unavailable (13, 14).
This rare mutation, p.S339F, is in exon 3 of the MEFV gene. This exon encodes the B-box domain, which interacts intramolecularly with the PYD (pyrin domain) to maintain pyrin in a repressed state. Therefore, it can be assumed that mutations in this region change the auto-inhibitory role of the domain (15). In the study by Stella et al. (13), they confirmed the pathogenicity of this mutation using different in silico tools. However, we confirmed, for the first time, its pathogenicity using the I-Mutant site. The I-Mutant website indicates that this substitution mutation alters the protein's free energy, thereby causing instability in its structure (16).
We identified an uncommon mutation, p.R202Q, in seven of 16 patients (43.75%). The p.R202Q variant was initially reported as a polymorphism (17). A close association was identified between this variation and FMF disease in the following years, which can be regarded as a mutation. Recent studies have shown its importance as a risk factor for the development of amyloidosis (18, 19). In the studies of Altamimi et al. in 2023 (n=132) and Capraz et al. in 2023 (n=1570) from Turkey, the most prevalent mutation was p.R202Q, with a frequency of 53.8% and 43.5%, respectively (20, 21). Although the p.R202Q mutation is uncommon in Iran (9), it was the second most prevalent mutation in the present study, with an allelic frequency of 43.75%. This mutation was observed in the compound heterozygous state (combination with p.E148Q, p.M694V, and p.V726A mutations) and complex genotype (p.M694V/p.M694I/p.R202Q). This rare complex genotype (p.M694V/p.M694I/p.R202Q) had not previously been reported in Iranian studies but was reported by Arpaci et al. in 2021 from Turkey (18).
Similar to our results, studies conducted by Beheshtian et al. in 2016 (n=951) and Farahzadi et al. in 2021 (n=252) identified genotypes p.R202Q/p.E148Q and p.M694V/p.R202Q in suspected FMF patients (3, 22). In a recent study by Nourbakhsh et al. in the southwest of Iran (n=13), p.R202Q was the most prevalent mutation, with a frequency of 38.46% (23). Differences in the frequency of the p.R202Q mutation in different populations, including Iran, might arise from socio-demographic characteristics, ethnic differences (considering the multiethnic nature of the Iranian population), and the number of studied patients. These results revealed that further studies are necessary to understand the prevalence and implications of the p.R202Q mutation in Iran.
To date, five mutations (p.M694V, p.M694L, p.M694del, p.M694I, and p.M694K) have been detected at codon 694 of the MEFV gene (Infevers, 2024). The methionine residues in codons 694 and 680 play a critical role in the function of pyrin. Thus, mutations in these codons can cause severe symptoms. In particular, the p.M694V mutation causes the most complicated form of FMF, and its homozygosity penetration is considerably high (99%) (24). As expected, according to the Tel-Hashomer criteria, our patients with mutations in these codons had a severe phenotype.
The pattern of inheritance for FMF is autosomal recessive, with a dominant inheritance hypothesis supported by numerous studies (OMIM 134610). P10 showed this pattern in our research. Although the whole gene was analyzed, we found only one mutation in this patient. This patient had a p.M694I (c.2040G>A) mutation and suffered from typical symptoms of FMF. Some studies have reported a dominant inheritance pattern for this mutation (25, 26).
Similar to other studies (6, 27), in spite of the clinical diagnosis, no mutation was observed in the coding and splicing regions of the MEFV gene in 24 out of the 40 studied patients. Genetic factors influence the occurrence of FMF, but it is also susceptible to various other influences, such as mutations in other auto-inflammatory genes, epigenetic or post-translational changes, environmental factors, and hidden biomarkers (28). It is important to note that PFAPA syndrome can sometimes present with FMF-like symptoms, and these patients respond to colchicine (29, 30). This potential overlap underscores the need for careful and thorough differential diagnosis. Meanwhile, this study did not cover the MEFV gene intronic regions.
FMF diagnosis is based on clinical manifestations as well as genetic testing and is supported by ethnic origin and family history. All parents of the patients in whom the mutation was detected had consanguineous marriages. Specifically, 37.5% were first cousins ​​ and the rest were distant relatives. In 43.75% of patients, a positive family history of FMF was present, particularly among close relatives. In the 2019 study by Rostamizadeh et al. on Azari-Turkish FMF patients, the ratio of parenteral consanguinity and family history of FMF was found to be 24.2% and 20%, respectively (6).
Regarding the clinical presentation of FMF, fever and abdominal pain are the most common complaints for all ethnicities (31). The frequency of clinical manifestations varies across populations. In the present study, the most prevalent clinical symptoms of the patients were fever and abdominal pain, consistent with findings in most other studies (6,12). Vomiting, myalgia, joint pain, and chest pain were other common symptoms. Unlike many studies, no signs of skin rash were observed in our patients (20, 32). A less common clinical finding was febrile seizures observed in P3 (with genotype p.V726A/p. E148Q). Studies have reported that the frequency of febrile seizures grows in patients with FMF compared to the general population (33, 34). This intriguing finding, which may be due to high fever during FMF attacks in individuals predisposed to febrile seizures or could be a neurologic complication of FMF, opens up new avenues for research and potential implications for patient care.

 

Conclusion

These findings helped to identify mutational hotspots in the population, discover novel or rare mutations, and design a local molecular diagnostic strategy. Such research ensures faster and easier identification of patients and carriers, expediting treatment and preventing long-term complications. In this way, our study further confirmed the pathogenicity of the rare p.S339F mutation. In addition, in line with previous research from different populations, this study suggested the high frequency of potentially pathogenic mutation of p.R202Q. Besides, its association with the full spectrum of FMF features underscores the importance of including this mutation in the routine molecular screening of patients. Further studies with a larger sample size are needed to discover other MEFV gene mutations, confirm their clinical significance, and detect hotspot regions in different populations.

 

Declarations

Acknowledgements

This article is the result of Ms. Zohreh Keykhodaei's thesis, which she completed to earn a master's degree in Immunology from AJUMS, with support from the Cellular and Molecular Research Center, Ahvaz Jundishapur University of Medical Sciences (CMRC-9902). We appreciate the cooperation and trust of the patients and their families, as well as that of Ms. Atefeh Heydari.

 

Ethical Considerations

Under the Ethics Committee of Ahvaz Jundishapur University of Medical Sciences, the study was approved (Approval ID: IRAJUMS.MEDICINE.REC.1399.003). All participants signed an informed consent form following detailed explanations of the study's objectives and procedures.

 

Authors' Contributions

Project administration, P.G.; design of experiments, A.G.; supervised patients' clinical studies, M.F., A.K., and A.S.; planning and conducting experiments, P.G., Z.K.; writing—original draft preparation, Z.K.; editing, P.G.

 

Conflicts of Interest

The authors declare that there are no conflicts of interest.

 

Fund or Financial Support

Ahvaz Jundishapur University Medical Sciences funded this study (CMRC-9902).

 

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