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Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Objective

Familial Mediterranean fever (FMF) is an autosomal-recessive disorder characterized by recurrent attacks of fever, with abdominal, thoracic, or articular pain. FMF is particularly common in Mediterranean populations, while other populations are rarely affected. MEFV gene analysis provides the only objective diagnostic criterion for FMF. However, the spectrum of MEFV mutations, which was first established in classically affected populations, remains insufficiently studied in other populations. The purpose of this study was to assess involvement of MEFV in the phenotype of western European Caucasian patients with a clinical diagnosis of FMF.

Methods

Mutation analysis was performed in 208 Caucasian patients from western Europe, by screening for the most common MEFV mutations in exons 2, 3, 5, and 10, and by sequencing the promoter region and the whole MEFV coding sequence in 21 of these patients.

Results

None of the patients carried 2 mutated alleles. Only 2 patients carried 1 mutated allele.

Conclusion

FMF-like syndromes in western European Caucasian populations cannot be explained by MEFV mutations. These results should be helpful in avoiding laborious and costly MEFV molecular analyses that, at the population level, seem to be of poor diagnostic value in the case of western European Caucasian patients, and rather should prompt a search for other causes in those patients.

Familial Mediterranean fever (FMF) is the most common of the hereditary recurrent fevers. FMF is characterized by recurrent attacks of fever accompanied by abdominal, thoracic, or articular pain. This autosomal recessive disease (MIM no. 249100) commonly affects 4 Mediterranean populations: subjects of Armenian, Arab, Sephardic Jewish, and Turkish extraction. The diagnosis of FMF is based on 2 sets of clinical criteria (those described by Livneh et al [1] and the Tel-Hashomer criteria [2]); finding 2 mutated MEFV alleles provides objective support for a clinical diagnosis of FMF. The spectrum of MEFV gene mutations has been well established within the 4 classically affected populations but remains to be clearly defined in other populations. The aim of this study was to investigate involvement of the MEFV gene in western European Caucasian patients presenting with a disease phenotype compatible with FMF.

PATIENTS AND METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

Patients.

The MEFV gene was studied in 208 western European Caucasian patients living in France, none of whom were of known Mediterranean origin (Table 1). Phenotype features were recorded by physicians using a standardized clinical form, and the diagnosis of FMF was established according to accepted clinical criteria (1). Informed consent was obtained from patients, according to the recommendations of French ethics committees.

Table 1. MEFV mutations in 208 European Caucasian patients
 French (n = 142)*European (n = 66)
  • *

    Both parents are French with no known Mediterranean origin.

  • Parents are of western and/or central European origin (e.g., Belgian, Czech, Dutch, French, German, Polish, Portuguese, Romanian, or Swiss).

Mutated alleles20
c.1772T>C (p.I591T)10
c.2084A>G (p.K695R)10

Mutation analysis.

In 116 patients, mutation screening was performed by denaturing gradient gel electrophoresis (DGGE) of exons 2, 3, 5, and 10 of MEFV, as previously described (3). The next 92 consecutive patients were studied by using a reverse hybridization assay (FMF StripAssay; ViennaLab Labordiagnostika, Vienna, Austria) that allows detection of the 12 most frequent MEFV mutations located in exon 2 (p.E148Q [c.442G>C]), exon 3 (p.P369S [c.1105C>T]), exon 5 (p.F479L [c.1437C>G]), and exon 10 (p.M680I [c.2040G>C], p.M680I [c.2040G>A], I692del [c.2076_2078del], p.M694V [c.2080A>G], p.M694I [c.2082 G>A], p.K695R [c.2084A>G], p.V726A [c.2177T>C], p.A744S [c.2230G>T], and p.R761H [c.2282G>A]). This test detects 99.3% of the mutations characterized by DGGE analysis in patients from commonly affected populations; since its clinical evaluation and validation (4), it has been used routinely in our laboratory. All mutations were confirmed by genomic DNA sequencing.

An additional mutation analysis was performed in 21 of these patients (see Results for details) by sequencing the −784 promoter region, the 10 MEFV exons, and the exon–intron boundaries. Twenty of these patients were selected because their disease phenotype satisfied the more restrictive Tel-Hashomer clinical criteria (2). The additional individual is the patient in whom we identified a MEFV mutation in the heterozygous state; we therefore checked the whole coding region to search for a mutation on the second MEFV allele.

Oligonucleotide sequences for polymerase chain reaction and sequencing were as follows: promoter, 5′-TCCAGCATCTTGGCACACTGT-3′ and 5′-TGAGCAAGAAAAGGCAGGTTGT-3′; exon 1, 5′-ACAACCTGCCTTTTCTTGCTCA-3′ and 5′-AGCTCCTGGTCCCCTTTCCC-3′; exon 2, 5′-CCTCAATTTCTAAACTTTAATATCC-3′ and 5′-CGTGCCCGGCCGAGCCATTC-3′; exon 3, 5′-TCTGTGTAAGCAACTTGGGTTTG-3′ and 5′-TTGGGAAAATGAAGTAAGGCCC-3′; exon 4, 5′-CTAAAGATGGCAGGAGCCAA-3′ and 5′-CGGGGACCCCTGCTCACT-3′; exon 5, 5′-CTGGGGGTTCCTGGACATCC-3′ and 5′-GAGCTGGGAGCCTGAGGCAT-3′; exon 6, 5′-CAGGGAGCCCTGGTAAGTGC-3′ and 5′-CAGGTCCCTTCTGGGACTGTC-3′; exons 7 and 8, 5′-GCCACCCCCTCGCTCCGG-3′ and 5′ACAAGGGAACACTGCAACAACC-3′; exon 9, 5′-CCTCTTAATGCTAATGCTCAGG-3′ and 5′-TTGGGGTAAGCGGTTTCTGCA-3′; exon 10, 5′-ATTTTCTTTGCAGTTAATGTGATTC-3′ and 5′-GATACAAGGCCAGAAGCAGG-3′.

RESULTS

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We analyzed the genotype of 208 western European Caucasian patients living in France, all of whom had a clinical diagnosis of FMF according to commonly used clinical criteria (1). The patients presented with recurrent attacks of fever accompanied by abdominal pain (70.7% of patients) and/or articular pain (43.8%) and/or thoracic pain (19.7%); 12.5% of the patients had only recurrent attacks of fever (Table 2). Molecular testing for the most frequent MEFV mutations situated in exons 2, 3, 5, and 10 of MEFV detected only 1 individual bearing a single p.K695R mutated allele (Table 1), and 2 individuals carrying the p.E148Q variant. We then performed an additional analysis of the MEFV gene (including the promoter region and the whole MEFV coding sequence) by genomic DNA sequencing in the patient carrying the p.K695R mutation. No other mutation was identified.

Table 2. Clinical manifestations of the patients
SymptomFrequency, %
Recurrent febrile attacks, accompanied by: 
 Abdominal pain70.7
 Articular pain43.8
 Thoracic pain19.7
 Fever alone12.5
Erysipelas-like erythema12
AA amyloidosis without predisposing disease1.4

All 208 patients satisfied the criteria described by Livneh et al (1); among them, 36 had a disease phenotype that also met the more restrictive Tel-Hashomer clinical criteria (2). These data reflect the fact that the Tel-Hashomer criteria set is not sensitive enough but is more specific than the Livneh criteria (Tchernitchko D, Amselem S: unpublished observations). Therefore, from among the 208 patients, we selected 20 patients who satisfied the more restrictive Tel-Hashomer clinical criteria set. In these latter patients, the analysis of the promoter and of the whole MEFV coding sequence led to the identification of only 1 patient who carried a p.I591T (c.1772T>C) sequence variation in the heterozygous state; this nucleotide variation is located in exon 9, a sequence that is not explored by DGGE or reverse dot methods.

DISCUSSION

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

The aim of this study was to test whether the MEFV gene is implicated in recurrent fevers similar to FMF among western European Caucasian patients with no known Mediterranean origin. Since identification of the MEFV gene, the spectrum of mutations has been established in populations commonly affected by FMF, such as Armenians, Arabs, Sephardic Jews, and Turks (5, 6), revealing an apparent mutational “hot spot” in exon 10. Thus, p.M694V, p.V726A, p.M680I, and p.M694I represent >90% of mutated alleles. Haplotype analysis has demonstrated that most FMF chromosomes descend from a common ancestor, in keeping with a founder effect (5, 6). Each of the mutations other than those described above are found in <1% of FMF chromosomes; some of them are isolated mutations and are found especially in individuals who do not belong to commonly affected populations. However, in the absence of any functional test, the deleterious character of those mutations is difficult to assess. Furthermore, the FMF founder haplotypes were not established in populations other than the 4 classically affected populations (5, 6).

MEFV mutations have also been reported in Greeks and Italians (7, 8); however, those studies mentioned allele frequencies and/or geographic origins of the patients, and did not always describe their genotypes and ethnic origins. Moreover, the frequency of mutated alleles may be overestimated in those populations because of the high frequency of the p.E148Q variation; indeed, the involvement of p.E148Q in FMF is controversial (9–12).

The frequency of MEFV mutations in healthy European Caucasian populations is, to date, unknown. Our results show an extremely low frequency of common MEFV mutations in European Caucasian patients (the mutated allele frequency was 0.5%). Thus, in this population the probability of identifying a patient with 2 mutated alleles is dramatically weak. Two hypotheses might explain these observations. First, the molecular screening protocol may have failed to detect the mutations responsible for the phenotype of the patients. However, the MEFV gene analysis that included the promoter and the whole coding sequence also failed to identify mutations in both alleles in 21 of our patients. Second, the MEFV gene may not be a key candidate for recurrent fevers among western European Caucasian patients. In this regard, it is important to emphasize that the clinical criteria established in classically affected populations (1, 2) do not allow discrimination between FMF due to MEFV mutations, FMF unrelated to MEFV (13), or other familial recurrent fevers. In addition, nonhereditary fevers may be implicated in the observed phenotype. In this regard, a recent study of febrile illness of unknown origin (14) showed that the cause of the fever remained obscure in >50% of patients.

In conclusion, our results strongly suggest that MEFV gene mutations are not commonly involved in FMF-like clinical manifestations in western European Caucasians populations. These data, therefore, should be helpful in avoiding laborious and costly MEFV molecular analyses that, at the population level, seem to be of poor relevance in western European Caucasian patients with a recurrent fever. Instead, the results should prompt a search for other causes in such patients. It would be of particular interest to test whether a similar situation is encountered among non-Mediterranean patients of other origins.

Acknowledgements

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES

We thank Ray Horn for critical reading of the manuscript.

REFERENCES

  1. Top of page
  2. Abstract
  3. PATIENTS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. Acknowledgements
  7. REFERENCES
  • 1
    Livneh A, Langevitz P, Zemer D, Zaks N, Kees S, Lidar T, et al. Criteria for the diagnosis of familial Mediterranean fever. Arthritis Rheum 1997; 40: 187985.
  • 2
    Pras M. Familial Mediterranean fever: from the clinical syndrome to the cloning of the pyrin gene. Scand J Rheumatol 1998; 27: 927.
  • 3
    Cazeneuve C, Sarkisian T, Pecheux C, Dervichian M, Nedelec B, Reinert P, et al. MEFV-gene analysis in Armenian patients with familial Mediterranean fever: diagnostic value and unfavorable renal prognosis of the M694V homozygous genotype-genetic and therapeutic implications. Am J Hum Genet 1999; 65: 8897.
  • 4
    Tchernitchko D, Legendre M, Delahaye A, Cazeneuve C, Niel F, Goossens M, et al. Clinical evaluation of a reverse hybridization assay for the molecular detection of twelve MEFV gene mutations. Clin Chem 2003; 49: 19425.
  • 5
    The International FMF Consortium. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. Cell 1997; 90: 797807.
  • 6
    The French FMF Consortium. A candidate gene for familial Mediterranean fever. Nat Genet 1997; 17: 2531.
  • 7
    Konstantopoulos K, Kanta A, Deltas C, Atamian V, Mavrogianni D, Tzioufas AG, et al. Familial Mediterranean fever associated pyrin mutations in Greece. Ann Rheum Dis 2003; 62: 47981.
  • 8
    La Regina M, Nucera G, Diaco M, Procopio A, Gasbarrini G, Notarnicola C, et al. Familial Mediterranean fever is no longer a rare disease in Italy. Eur J Hum Genet 2003; 11: 550.
  • 9
    Mimouni A, Magal N, Stoffman N, Shohat T, Minasian A, Krasnov M, et al. Familial Mediterranean fever: effects of genotype and ethnicity on inflammatory attacks and amyloidosis. Pediatrics 2000; 105: E70.
  • 10
    Ben-Chetrit E, Lerer I, Malamud E, Domingo C, Abeliovich D. The E148Q mutation in the MEFV gene: is it a disease-causing mutation or a sequence variant? Hum Mutat 2000; 15: 3856.
  • 11
    Tchernitchko D, Legendre M, Cazeneuve C, Delahaye A, Niel F, Amselem S. The E148Q MEFV allele is not implicated in the development of familial Mediterranean fever. Hum Mutat 2003; 22: 33940.
    Direct Link:
  • 12
    Amselem S, Tchernitchko D, Cazeneuve C. E148Q MEFV sequence variation and familial Mediterranean fever. Ann Rheum Dis 2004. E-pub ahead of print.
  • 13
    Cazeneuve C, Hovannesyan Z, Genevieve D, Hayrapetyan H, Papin S, Girodon-Boulandet E, et al. Familial Mediterranean fever among patients from Karabakh and the diagnostic value of MEFV gene analysis in all classically affected populations. Arthritis Rheum 2003; 48: 232431.
  • 14
    Vanderschueren S, Knockaert D, Adriaenssens T, Demey W, Durnez A, Blockmans D, et al. From prolonged febrile illness to fever of unknown origin: the challenge continues. Arch Intern Med 2003; 163: 103341.