MINIREVIEW

Molecular Genetic Testing for Familial Mediterranean Fever

Milhan Telatar and Wayne W. Grody1

Divisions of Molecular Pathology and Medical Genetics, Departments of Pathology & Laboratory Medicine and Pediatrics,

Molecular Genetics and Metabolism 71, 256–260 (2000)doi:10.1006/mgme.2000.3047, available online at http://www.idealibrary.com on

UCLA School of Medicine, Los Angeles, California 90095-1732

July 2

Received

Familial Mediterranean fever (FMF) is an autoso-mal recessive inflammatory disease primarily affect-ing populations of the Mediterranean basin. Be-cause of its remitting and relapsing nature overmany years, characterized by nonspecific symptomsthat resemble those of other, more common medicaland surgical conditions, many patients go undiag-nosed for long periods during which they are sub-jected to unnecessary, unrevealing, and sometimesrisky diagnostic and exploratory procedures. It isonly with the recent discovery of the causative geneand the advent of direct mutation testing that spe-cific laboratory diagnosis of FMF has become possi-ble. This article will present the current state of theart and knowledge about molecular genetic testingfor this condition, and the rationale behind it.

POPULATION GENETICS

Although the disease has been reported in a vari-ety of ethnic groups, the majority of cases continueto occur in four Mediterranean populations: Arabs,Armenians, Turks, and Sephardic Jews (1). The car-rier frequency has been estimated to be as high as 1in 6.4 in North African Jews from Morocco, Algeria,and Tunisia, 1 in 5–10 in Ashkenazi Jews, and 1 in7 in Armenian–Americans (2,3).

CLINICAL FEATURES

The disease typically presents as recurrent, short(lasting 2–4 days), self-limiting episodes of fever

1 To whom correspondence should be addressed. Fax: (310)206-5178. E-mail: wgrody@mednet.ucla.edu.

2561096-7192/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

8, 2000

accompanied by abdominal, chest, or joint pain, de-pending on the serous membrane involved. The at-tacks thus appear as peritonitis, pleuritis, or syno-vitis, which recur at irregular intervals and inunpredictable sequence. Peritoneal attacks are ac-companied by fever and abdominal pain from a vari-able point of origin, and last 6–12 h (1). While 95%of the patients eventually suffer a peritoneal attack,for only 50% is it the first type of episode experi-enced. Pleural attacks are accompanied by chestpain and occur in up to 50% of patients. The pleurisyis usually unilateral, with diminished breath soundson the affected side. Synovial attacks are the secondmost common manifestation of FMF and may re-solve within a month or persist for as long as a year.Arthralgia and arthritis occur in 75% of North Afri-can Jewish FMF patients. Erysipelas-like erythem-atous patches are characteristic of the cutaneouslesions in these patients, with up to 46% so affected.Warm and tender lesions that cover an area of10–15 cm in diameter appear on the skin of thelower extremities below the knee, on the dorsum ofthe foot, or in the ankle area.

The most severe complication of FMF is amyloid-osis that results in nephrotic syndrome and ulti-mately leads to terminal renal failure if not treated.Widespread tissue deposition of serum amyloid(SAA), an acute phase protein, has been character-ized as a determinant of amyloidosis in FMF pa-tients, especially in the kidneys (4). There is no

relation between the severity of the inflammatorycrisis and SAA level which is elevated during bothinflammatory attacks and attack-free intervals. If

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initiated before renal function is impaired, daily andlifelong administration of colchicine amelioratesboth the inflammatory attacks and amyloidosis de-velopment (5). The clinical course and manifesta-tions of the disease, including fever, peritonitis,pleurisy, rashes, and amyloidosis, are highly vari-able in their pattern, frequency, intensity, and age ofonset. Sixty percent of FMF patients experiencetheir first attack before the age of 10, 90% beforereaching age 20, and the rest before 40. Late onset ofFMF after 40 years of age apparently represents aclinically distinct, milder form of the disease (6).

General clinical laboratory findings in the disor-der are either normal or nonspecific. Especially dur-ing attacks, there may be granulocytosis of about15,000–30,000/mL and elevated erythrocyte sedi-mentation rate (7).

MOLECULAR GENETICSAfter a 10-year effort, the gene for FMF (MEFV)

was positionally cloned from a 115-kb candidate in-terval on chromosome 16p13 by two independentconsortia in 1977 (8–10). The gene spans approxi-mately 10 kb and is composed of 10 exons whichgenerate a 3.7-kb transcript (11). The 781-amino-acid protein has been called marenostrin by theFrench FMF Consortium (8) and pyrin by the Inter-national FMF Consortium (9). It is expressed in

TAReported M

No. Nucleotide Amino acid Exon

1 442G 3 C E148Q 2 Berno2 501G 3 C E167D 2 Berno3 800C 3 A T267I 2 Berno4 1105C 3 T P369S 3 Aksen5 1223G 3 A R408Q 3 Cazen6 1437C 3 G F479L 5 Berno7 2040G 3 C M680I 10 Intern8 2040G 3 A M680I 10 Aksen9 2042C 3 T T681I 10 Booth

10 2076-2078del I692del 10 Berno11 2078-2080del M694del 10 Booth12 2080A 3 G M694V 10 Intern13 2082G 3 A M694I 10 Frenc14 2084A 3 G K695R 10 Berno15 2177T 3 C V726A 10 Intern16 2230G 3 T A744S 10 Berno17 2282G 3 A R761H 10 Berno

GENETIC TESTING FOR FAM

mature neutrophils, suggesting that it functions asan inflammatory regulator. It is a new member of afamily of highly conserved genes that includes nu-

clear effector molecules like RO52 autoantigen andnucleic acid binding proteins that regulate inflam-mation (12).

Since the cloning of the gene, 17 mutations havebeen identified and tested in different ethnic groups(Table 1). Four missense mutations (M694V, M694I,V726A, and M680I) in exon 10 (two of which involvethe same codon) were responsible for a large propor-tion of mutations observed in the two initial studies(8,9). Each of these mutations was associated withdistinct haplotypes. M694V was observed in a largepercentage of carrier chromosomes in families ofvarious ethnic origins (North African Jews, IraqiJews, Armenians, Arabs, and Turks) in associationwith one specific haplotype, MED. The M694I mu-tation was associated with Arab haplotype ARA2,while the V726A mutation with haplotype ARM3/Druze was found in Armenians, Druze, and Iraqiand Ashkenazi Jews. Mutation M680I was observedonly in Armenians on a distinct Armenian haplo-type, ARM2 (13).

The observation of the M694V mutation on fourdifferent haplotypes that are identical at a series ofintragenic single nucleotide polymorphisms (SNPs)indicates an ancient founder effect for the origin of alarge fraction of FMF cases from the Mediterraneanbasin. Furthermore, the fact that mutations M694Iand V726A were seen in association with a specific

1Mutations

Reference

, 1998 (11), 1998 (11), 1998 (11)h et al., 1999 (14)t al., 1999 (13), 1998 (11)l FMF Consortium, 1997 (9); French FMF Consortium, 1997 (8)h et al., 1999 (14)1998 (24), 1998 (11)1998 (24)l FMF Consortium, 1997 (9); French FMF Consortium, 1997 (8)Consortium, 1997 (8)

, 1998 (11)l FMF Consortium, 1997 (9); French FMF Consortium, 1997 (8), 1998 (11), 1998 (11)

257L MEDITERRANEAN FEVER

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haplotype in populations that have been separatedfor many centuries demonstrates that these muta-tions are likely to date back at least to biblical times.

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Mutation E148Q in exon 2 was found to be thesecond most common mutation occurring in patientsof several ethnicities with different haplotypes(11,14). The recent observation that two of thehealthy parents were homozygous for the E148Qmutation and that in two parents this mutation wasnot associated with the FMF-bearing allele raisesthe question of whether E148Q is a disease-causingmutation with low penetrance or a benign sequencevariant with no pathological consequences (15). Itappears to be of low risk unless coupled with themore uniformly severe mutation M694V. The situa-tion is thus somewhat reminiscent of the commonyet low-penetrant mutation H63D in the HFE geneof hereditary hemochromatosis, which is primarilyof concern only when it is found in compound het-erozygous form with the major pathologic alleleC282Y (16). P369S and K695R are two other muta-tions with low penetrance which, together withE148Q, account for the high carrier frequency butrather low clinical prevalence in the Ashkenazi Jew-ish population (14,17).

Booth et al. (18) showed evidence for autosomalominant inheritance of FMF with certain geno-ypes in five families. Dominant FMF was associ-ted with E148Q/M694I carried on the same allelen two independent Indian and Turkish families, orith heterozygosity for an M694del mutation in

hree unrelated British families. Complete MEFVequencing failed to identify any coding region ab-ormality in the other allele in any of these cases.No consistent correlations have been found be-

ween particular mutations and the type or the se-erity of FMF symptoms. The sole exception seemso be a significant association between amyloidosisnd the M694V mutation (19–21). Amyloidosis wasresent in 44 of 171 M694V homozygotes (25.7%), in2 of 143 compound heterozygotes (15.4%), and in 7f the 57 patients carrying other mutations (12.3%)19). Aside from that association, however, specific

EFV mutations are not the sole determinants ofhenotypic features such as age of onset, frequencyr duration of attacks, expression of particularymptoms, or response to colchicine. It appears thatome unknown environmental factors or modifyingenes act as accomplices in this disease (7). Theombinations of clinical manifestations among FMFatients are quite heterogeneous and none of thethnic populations has a range of symptoms that

258 TELATAR

istinguishes one from another. Furthermore, FMFhares many clinical features with the recessivelynherited hyperimmunoglobulinemia D and periodic

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fever syndromes, and there are many dominantlyinherited periodic fever syndromes that might bemistaken for FMF (22,23). These findings emphasizethe importance of performing molecular analysis onall suspected FMF patients.

DNA TESTING

A number of laboratories are now offering molec-ular genetic testing for FMF using simple PCR-based methods such as amplified restriction frag-ment length polymorphism, amplification refractorymutation system, allele-specific oligonucleotideprobe hybridization, or DNA sequencing. Thesemethods capitalize on the chance disruption or cre-ation of a restriction endonuclease recognition siteby a mutation, the differential amplification or hy-bridization of gene regions using allele-specific PCRprimers or probes, or direct identification of nucleo-tide changes by DNA sequencing. The latter methodhas the potential advantage of detecting rare orunknown mutations beyond those in the standardscreening panel. For example, during routine diag-nostic testing in our laboratory using this method,we detected a novel three-nucleotide substitution,

FIG. 1. Detection of a novel MEFV mutation in exon 10 by DNAsequence analysis.The sequencing ladder reveals heterozygosity forthe three-nucleotide substitutions (593 39) GCA3 CAT at position2183. This change results in two amino acid substitutions involvingadjacent codons (S728T, I729F). The patient is also (compound)heterozygous for the common V726A mutation (GTT 3 GCT) seentwo codons away on the same autoradiogram.

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2183GCA3 CAT (causing missense amino acid sub-titutions in two adjacent codons, S728T and I729F)Fig. 1). The patient was symptomatic, with muta-

ILIA

tion V726A carried on the other allele. On the otherhand, as is well known from BRCA1/2 gene testing,DNA sequencing has the disadvantage of picking upboth benign polymorphisms and nucleotide changesof uncertain significance. Sometimes the dispositionof these findings will be obvious, as in the case of asilent valine-to-valine substitution we detected atamino acid position 722 (2166G3 A) in one patient;at other times, in the absence of previous reports inpatients or large numbers of normal controls, theclinical significance will not be so clear-cut.

Molecular testing provides an accurate diagnosisof FMF in patients presenting with suspicious clin-ical symptoms, enables early detection of carriers fortimely institution of colchicine therapy, and allowsassessment of the risk for amyloidosis later in life.Ethnic background is an important predictor of find-ing one of the reported mutations, since patients ofhigh-risk background were shown to carry MEFVmutations even when the clinical features wereatypical (7). In other ethnic groups, on the otherhand, the lack of a comprehensive spectrum ofknown FMF mutations limits the power of molecu-lar testing for differential diagnosis. Even in symp-tomatic patients from ostensibly high-risk ethnicbackgrounds, it has not been uncommon in our ex-perience to detect only a single mutant allele. Insuch cases we usually accept the DNA finding assupportive of the FMF diagnosis, provided the req-uisite clinical criteria are present, and assume thatthe second mutation is not detected due to rarity orethnic admixture. It is hoped that continued study ofthe MEFV gene will reveal additional mutations ofclinical relevance, as well as more specific and effec-tive gene- or protein-targeted therapies for this im-portant disorder.

REFERENCES

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3. Rogers DB, Shohat M, Petersen GM, Bickal J, Congleton J,Schwabe AD, Rotter JI. Familial Mediterranean fever inArmenians: Autosomal recessive inheritance with high genefrequency. Am J Med Genet 34:168–172, 1989.

GENETIC TESTING FOR FAM

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5. Cabili S, Zemer D, Pras M, Aviram A, Sohar E, Gafni J. Theprevention of amyloidosis in familial Mediterranean feverwith colchicine. Proc Eur Dial Transplant Assoc Eur RenAssoc 21:709–811, 1985.

6. Tamir N, Langevitz P, Zemer D, Pras E, Shinar Y, Padeh S,Zaks N, Pras M, Livneh A. Late-onset familial Mediterra-nean fever (FMF): A subset with distinct clinical, demo-graphic, and molecular genetic characteristics. Am J MedGenet 87:30–35, 1999.

7. Samuels J, Aksentijevich I, Torosyan Y, Centola M, Deng Z,Sood R, Kastner DL. Familial Mediterranean fever at themillennium: Clinical spectrum, ancient mutations, and asurvey of 100 American referrals to the National Institutesof Health. Medicine 77:268–297, 1998.

8. French FMF Consortium. A candidate gene for familial Med-iterranean fever. Nature Genet 17:25–31, 1997.

9. International FMF Consortium. Ancient missense muta-tions in a new member of the RoRet gene family are likely tocause familial Mediterranean fever. Cell 90:797–807, 1997.

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13. Cazeneuve C, Sarkisian T, Pecheux C, et al. MEFV-geneanalysis in Armenian patients with familial Mediterraneanfever: Diagnostic value and unfavourable renal prognosis ofthe M694V homozygous genotype—Genetic and therapeuticimplications. Am J Hum Genet 65:88–97, 1999.

14. Aksentijevich I, Torosyan Y, Samuels J, et al. Mutation andhaplotype studies of familial Mediterranean fever revealnew ancestral relationships and evidence for a high carrierfrequency with reduced penetrance in the Ashkenazi Jewishpopulation. Am J Hum Genet 64:949–962, 1999.

15. Ben-Chetrit E, Lerer I, Malamud E, Domingo C, AbeliovichD. The E148Q mutation in the MEFV gene: Is it a disease-causing mutation or a sequence variant? Hum Mutat 15:385–386, 2000.

16. Risch N. Haemochromatosis, HFE and genetic complexity.Nat Genet 17:375–376, 1997.

17. Pras M, Bronshpigel N, Zemer D, Gafni J. Variable inci-dence of amyloidosis in familial Mediterranean fever amongdifferent ethnic groups. Johns Hopkins Med J 150:22–26,1982.

18. Booth DR, Gillmore JD, Lachmann HJ, et al. The geneticbasis of autosomal dominant familial Mediterranean fever.QJM 93:217–221, 2000.

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19. Mimouni A, Magal N, Stoffman N, et al. Familial Mediter-ranean fever: Effects of genotype and ethnicity on inflam-matory attacks and amyloidosis. Pediatrics 105:E70, 2000.

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21. Livneh A, Langevitz P, Shinar Y, Zaks N, Kastner DL, Pras

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22. Drenth JP, Mariman EC, Van der Velde-Visser SD, RopersHH, Van der Meer JW. Location of the gene causing hyper-immunoglobulinemia D and periodic fever syndrome differsfrom that for familial Mediterranean fever: InternationalHyper-IgD Study Group. Hum Genet 94:616–620, 1994.

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