A severe autosomal-dominant periodic inflammatory disorder with renal AA amyloidosis and colchicine resistance associated to the MEFV H478Y variant in a Spanish kindred: An unusual familial Mediterranean fever phenotype or another MEFV-associated periodic inflammatory disorder?



Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurring short attacks of fever and serositis. Secondary AA amyloidosis is the worst complication of the disease and often determines the prognosis. The MEFV gene, on chromosome 16p13.3, is responsible for the disease and around 30 mutations have been reported to date. Colchicine is the standard FMF treatment today, and prevents both attacks and amyloid deposition in 95% of patients. Here we describe a three-generation Spanish kindred with five family members affected by a severe periodic inflammatory disorder associated with renal AA amyloidosis and colchicine unresponsiveness. Clinical diagnosis of definite FMF disease was made based on the Tel-Hashomer criteria set. Genetic analyses revealed that all subjects were heterozygous for the new H478Y MEFV variant, segregating with the disease. In addition, mutations in the TNFRSF1A and CIAS1/PYPAF1/NALP3 genes, related to the dominantly inherited autoinflammatory periodic syndromes, were ruled out. However, the dominant inheritance of the disease, the long fever episodes with a predominant joint involvement, and the resistance to colchicine in these patients raise the question of whether the periodic syndrome seen in this kindred is a true FMF disease with unusual manifestations or rather another MEFV-associated periodic syndrome. We conclude that the new H478Y MEFV mutation is the dominant pathological variant causing the inflammatory periodic syndrome in this kindred and that full-length analyses of the MEFV gene are needed to obtain an adequate diagnosis of patients with clinical suspicion of a hereditary periodic fever syndrome, especially those from non-ancestral populations. © 2003 Wiley-Liss, Inc.


The hereditary periodic fever (HPF) syndromes are a rare group of autoinflammatory syndromes of genetic basis characterized by recurrent episodes of fever and serosal inflammation, without infectious etiology. Familial Mediterranean fever (FMF), Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS) are recessively transmitted, whereas the TNF receptor-associated periodic syndrome (TRAPS) and familial urticarial syndromes—familial cold urticaria/familial cold autoinflammatory syndrome (FCU/FCAS) and Muckle–Wells syndrome (MWS)—are dominantly inherited. Tissue deposition of the acute phase protein serum amyloid A (SAA) may lead to the fatal development of secondary AA amyloidosis in some FMF and TRAPS patients. The diagnosis of these HPF syndromes is based on an accurate description of the medical history of the patient, including age of onset, duration and periodicity of attacks, main symptoms, uncommon manifestations and ethnic origin. Recently, several genes involved in HPF syndromes have been identified: the MEFV gene on chromosome 16p13.3 associated with FMF [The International FMF consortium, 1997; The French FMF consortium, 1997], the MVK gene on chromosome 12q24 associated with HIDS [Drenth et al., 1999; Houten et al., 1999], the TNFRSF1A gene on chromosome 12p13 associated with TRAPS [McDermott et al., 1999] and finally, the CIAS1/PYPAF1/NALP3 gene on chromosome 1q44 associated with the MWS and FCU/FCAS [Hoffman et al., 2001]. Genetic analyses in search of disease causing mutations often support the clinical diagnosis.

FMF, the most frequent HPF syndrome, is highly prevalent in Mediterranean and Middle Eastern populations: non-Ashkenazi Jews, Arabs, Turks, and Armenians [The International FMF consortium, 1997; The French FMF consortium, 1997]. However, it is also present in non-ancestral populations such as Greeks, Italians, Spanish, Portuguese [Touitou, 2001], British [Booth et al., 2000], and Belgians [Anonymous, 1999]. Occasionally, patients of Indian, Chinese, Afghan, and Hungarian origin have also been described [Bernot et al., 1998; Booth et al., 1998]. The disease is characterized by recurrent short (1–3 days) episodes of inflammation and serositis including fever, peritonitis, pleuritis, arthritis, and skin lesions [Sohar et al., 1967]. The cloning of the MEFV gene has provided a molecular approach to FMF diagnosis, which is particularly valuable in patients with clinical suspicion of the disease [Grateau et al., 2000; Nir-Paz et al., 2000]. Thirty-three mutations in the MEFV gene have been reported (Infevers data base http://fmf.igh.cnrs.fr/infevers), accounting for 80% of FMF chromosomes in the classical autosomal recessive pattern of inheritance. Recently, two cases of autosomal dominant transmission have been reported: the deletion of the M694 residue in two unrelated British families and the M694I-E148Q complex allele in two unrelated families of Turkish and Indian origin [Booth et al., 2000]. These mutations are considered to be solitary pathological variants.

Here we describe a three-generation Spanish kindred with a severe dominantly inherited periodic inflammatory disorder complicated with renal AA amyloidosis. This phenotype is associated with a new H478Y mutation in the MEFV gene, responsible for FMF disease. However, the long episode duration with predominant joint involvement, the dominant inheritance pattern and colchicine unresponsiveness in these patients raise the question that whether the disorder seen in this kindred is FMF disease with an unusual phenotype or a new MEFV-associated disorder. The lack of response to colchicine treatment prompted the use of biological TNF blocking therapy with infliximab, which has shown promising results elsewhere.


Patients and Control Subjects

Five patients from a three-generation Spanish family, suffering from a periodic inflammatory syndrome, and their healthy relatives (six individuals) were studied. Approval for the study was obtained from the Institutional Review Board of the Hospital Clínic, Barcelona, and written-informed consent was obtained from the patients. A panel of 100 ethnically matched healthy donors from our DNA bank were used.

Figure 1 shows the familial pedigree and the dominant inheritance in this kindred. Clinical features of each patient (Table I) were recorded through a standardized clinical questionnaire on HPFs. Fever episodes started at a similar age in all affected members, between the age of 9 and 13, with a symptom-free interval ranging from 6 weeks to 3 months. Periodic episodes consisted on high bouts of fever (39°C) with arthritis, arthralgias, and skin lesions resembling an erysipela-like erythema. Whereas fever episodes lasted from 3 to 6 days, arthralgia persisted for up to 3–9 days in a typical attack and exceptionally up to 15–25 days. The joints mainly involved were wrists, fingers, and tarsus, usually in polyarticular and migratory attacks. Patient no. 5, with a very severe form of the condition, was confined to a wheelchair. On several occasions, the patients complained of abdominal and thoracic pain secondary to serositis (peritonitis and pleuritis). All affected females were submitted to abdominal surgery for their clinical symptoms (sterile peritonitis in all cases). In some cases febrile attacks were related with stress situations, including cold, school examinations, and tiredness. No cases of deafness or urticaria have been described in this family. CRP was persistently high (>5 mg/dl, normal range <0.8 mg/dl) in all affected members, and increased significantly during attacks (15 mg/dl). ANAs and anti-tissular antibodies were negative in all family members. Traces of proteinuria were observed in patients no. 4 and 5. Renal amyloidosis, which developed in two affected females (no. 1 and 2) at the age of 50, started with proteinuria and progressive renal failure, reaching end stage renal disease (ESRD) 3 years later. Renal biopsy using immunohistochemical analysis confirmed the AA amyloidosis (Fig. 2). Interestingly, the clinical syndrome was much more severe in females than in the affected male (no. 3) in terms of periodicity and clinical manifestations (Table I).

Figure 1.

Autosomal dominant inheritance in the FMF Spanish kindred bearing the H478Y MEFV mutation. Shaded symbols represent affected family members, an unshaded symbols show non-affected individuals. NT, not tested.

Table I. Characteristics of all Affected Family Members of the FMF Spanish Kindred Bearing the H478Y MEFV Variant: Clinical Response to Colchicine and Infliximab Treatment, and Results of Molecular Analyses
CharacteristicsPatient no. 1Patient no. 2Patient no. 3Patient no. 4Patient no. 5
  • Numbers at the top row correspond to those of Figure 1.

  • *

    Unusual FMF clinical manifestations shown by the patients of this kindred. aAge deceased; bSinovitis; cWrisfs; finger and tarsus; polyarticular and migratory; dafter renal biopsy; eonly 3-month follow up; fCIAS1/PYPAF1/NALP3;

  • Yr, years; Wk, weeks; Dy, days; ESRD, end stage renal disease; NT, not tested; +/−, partial colchicine response.

Age (yr)60a49522423
Age of onset (yr)109131211
Attack frequency (wk)6–106–108–126–86–8
Attack durationb (dy)3–15*3–20*2–43–20*3–25*
Fever (°C)3939383939
Fever duration (dy)4–6*4–6*2–33–5*4–6*
Abdominal pain++++
Proteinuria (mgr/day)30003000<150150–300150–300
Renal amyloidosisd++
ESRD dialysis++
Age at amyloidosis (yr)50*48*
Colchicine response**+/−**
Infliximab responseNT+eNT++
MEFV genotypeNTH478Y/wtH478Y/wtH478Y/wtH478Y/wt
TNFRSF1A mutationsNT
CIAS1f mutationsNT
SAA1 genotypeNTα/ββ/βα/βα/β
Figure 2.

Renal amyloidosis with massive glomerular involvement. A: Congo red stain (×250). B: Immunohistochemical analysis with anti-SAA (×250). [The color figure can be viewed in the online issue, available at www.interscience.wiley.com.]

Colchicine was administered to all patients, at the highest tolerated doses (mean dose 1.5 mg/day; range: 1–2.5 mg/day) without success. Only the male member (no. 3) presented a partial response, with improvement in joint symptoms. Furthermore, in spite of the colchicine treatment the two older females (no. 1 and 2) developed renal amyloidosis and progressed to ESRD. Infliximab was administered to the two younger females (no. 4 and 5) after the approval of the Spanish Health Department. Before starting infliximab treatment, chest X-ray and PPD were performed to rule out an active pulmonary tuberculosis. Infliximab therapy was started with initial doses of 5 mg/kg/day, administered every 2 weeks during the first 2 months, subsequently every 4 weeks, and currently every 8 weeks. Colchicine was simultaneously administered during Infliximab treatment. Methotrexate was not indicated given the side effects of this drug and the poor general status of the patients.

Laboratory Studies

DNA/RNA extraction and cDNA synthesis

From whole blood samples, genomic DNA and total RNA were isolated using QIAamp DNA Blood Mini Kit (Qiagen, Germany) and Trizol reagent (Life Technologies, Gaitherburg, MD), respectively. Complementary DNA (cDNA) was synthesized by oligo (dT)12–18 priming (Life Technologies).

PCR amplification and sequencing

PCR conditions used for MEFV, TNFRSF1A CIAS1/PYPAF1/NALP3, and SAA1 genes had been described previously [The International FMF consortium, 1997; McDermott et al., 1999; Moriguchi et al., 1999; Hoffman et al., 2001]. Sequencing reactions (dRhodamine Terminator Cycle Sequencing Ready Reaction, PE Applied Biosystem, Warrington, Great Britain) were analyzed in an ABI 377 automated sequencer.

Soluble TNFR1 measurements

The commercially available ELISA kit (R & D Systems, Minneapolis) was used following the manufacturer's standard protocol.


Based on the Tel-Hashomer criteria for FMF diagnosis [Pras, 1998], all patients in the present kindred with the exception of the male patient no. 3 met one major criterion—recurrent febrile episodes accompanied by peritonitis, sinovitis or pleuritis—and two minor criteria—recurrent febrile episodes, erysipela-like erythema, or FMF in a first degree relative. Patient no. 3 met Livneh FMF diagnostic criteria [Livneh et al., 1997].

Genetic Findings

Molecular analyses consisting of screening for mutations in the entire coding sequence of the HPF syndromes related genes were performed. Comprehensive MEFV analyses were performed and a new variant, the H478Y MEFV mutation, caused by a C>T transition at nucleotide 1432 in exon 5, which results in a histidine-to-tyrosine exchange at codon 478, was found in the heterozygous state in all symptomatic members of this kindred (Fig. 3A); no other mutations were found after sequencing all 10 exons and intronic flanking regions of the MEFV gene. Additionally, none of the healthy members of this kindred were carriers of this mutation. The possibility that this new variant was a polymorphism was considered, but rejected after analyzing a panel of 100 ethnically matched control donors. Additionally, mutations in the genes responsible for all HPF syndromes with autosomal dominant inheritance were ruled out. No mutations were found in any of the 10 exons and flanking regions of the TNFRSF1A gene in any patient. Furthermore, normal serum levels of soluble TNFR1 were found in all, affected and unaffected, family members (data not shown). Sequencing of all nine exons and bounding sequences of the CIAS1/PYPAF1/NALP3 gene revealed no mutations.

Figure 3.

Electrophoregram of the new H478Y MEFV variant detected in both genomic (A) and complementary DNA (B). The C > T transition at nucleotide 1432 in exon 5 converts a histidine-to-tyrosine exchange in codon 478. Patient no. 2 in the present FMF kindred (top) and an unrelated healthy donor (bottom). [The color figure can be viewed in the online issue, available at www.interscience.wiley.com].

To demonstrate the dominant transmission of the H478Y MEFV variant in this kindred 10 polymorphic intragenic markers ranging from exon 2 to 9 of the MEFV locus were analyzed and haplotypes from both the mutated and the wild type MEFV allele for all patients were constructed. The SNPs used were: D102D C/T, G138G A/G, A165A A/C, and R202Q A/G in exon 2; R314R C/T in exon 3; the polymorphism i4 A/G in intron 4; E474E A/G, Q476Q G/A and D510D T/C in exon 5 and P588P A/G in exon 9. The H478Y mutation was found to be associated with the CGAGTGAGCG haplotype. This mutation-bearing haplotype was accompanied by a clearly distinct wild type haplotype in each patient (data not shown).

Additionally, RT-PCR expression analyses demonstrated that both the mutated and the normal allele of the MEFV gene were expressed in all patients (Fig. 3B).

Finally, none of the patients carried the amyloidosis-associated α/α genotypy of the MEFV-independent modifier factor SAA1 gene.

Treatment Response

TNF-blocking biological therapy was administered. After the third dose of Infliximab the clinical status of the two patients improved significantly. Patient no. 4 did not present a febrile syndrome during the whole year and patient no. 5 improved progressively and no longer needed a wheelchair. Though she presented two attacks during the year, they were shorter and less intense than before. CRP decreased progressively in both patients, especially in patient no. 4 who presents levels within the normal range. Infliximab tolerance was excellent in both patients, who now lead normal lives. After the initial positive experience with infliximab in patients no. 4 and 5, we decided to treat patient no. 2 who was receiving dialysis for renal amyloidosis. Initial response to infliximab was satisfactory, although the follow-up was too short to confirm the benefits of biological therapy in this patient. No specific side effects were observed.


We describe the clinical features of a Spanish kindred with a severe hereditary periodic inflammatory disorder.

All affected family members were clinically diagnosed with FMF disease on the basis of clinical criteria. Severity scores for each patient were notably high (mean score 9.5) according to Pras' FMF disease severity score [Pras, 1998].

Molecular analysis of the MEFV gene revealed that a new mutation H478Y, located in exon 5 of the gene, was present in the heterozygous state in all patients, segregating with the disease and absent in all living healthy family members. However, since (a) all patients showed episodes with longer fever and articular pain duration and a more severe joint involvement than expected in a classical FMF phenotype (Table I); (b) the dominant inheritance of this kindred does not fit the classical recessive pattern of FMF transmission; and (c) none of the patients showed a favorable response to continuous colchicine treatment, which is the other major FMF diagnostic criteria, we screened for mutations in the two genes responsible for the dominantly inherited HPF syndromes TRAPS and FCU/FCAS/MWS. However, no mutations were found in either the TNFRSF1A gene or the CIAS1/PYPAF1/NALP3 gene.

Few cases with dominant inheritance have been reported in FMF disease. All those described affected the M694 residue: the ΔM694 mutation, a trinucleotide deletion in one single allele, was observed in FMF patients of two unrelated British families and the M694I-E148Q complex allele in two FMF unrelated families of Turkish and Indian origin [Booth et al., 2000]. The existence of a second mutation in the MEFV promoter region or in another gene in the present kindred seems to be highly unlikely, since this would necessarily mean a high prevalence of these mutations in the Spanish population for co-inheritance (with the H478Y MEFV mutation) to occur in each of the three generations of this kindred. Furthermore, the dominant mode of inheritance of the H478Y mutation was demonstrated by analyzing a set of intragenic polymorphisms, which led to the identification of different non-mutated MEFV alleles in each patient. Finally, RT-PCR experiments were performed to study whether mutations in the MEFV promoter region would abrogate MEFV mRNA expression of the normal allele, revealing that both alleles were expressed in all patients. Pseudo-dominant inheritance has been described in certain populations where FMF is highly prevalent and marriages of a FMF patient to a MEFV carrier are frequent. However, this is not the case in the Spanish population [Touitou, 2001; and personal data]. All these data suggest that the H478Y mutation is the pathological variant responsible for the severe autosomal dominant periodic inflammatory phenotype seen in this kindred.

The H478Y MEFV variant produces a non-conservative substitution in a region comprising the coiled-coil domain of pyrin/marenostrin, the protein involved in FMF disease. This domain may be involved in protein–protein interactions [The International FMF consortium, 1997]. Thus, it is possible that the mutation would compromise pyrin function, either by impeding homodimerization with wild type pyrin or by abrogating interaction with other proteins. The full-length gene sequencing strategy adopted recently by some groups has increased the number of new MEFV mutations along the entire gene sequence [Bernot et al., 1998]. Most of them are private mutations found in non-ancestral populations that may also cause severe FMF disease [Touitou, 2001]. Recently, new SNPs (E474E, Q476Q, R501R, I506I, D510D) and true FMF causing mutations (E474K, F479L, V487M, R501G; Infevers data base) have been described in exon 5 suggesting the functional importance of this domain. Further studies are needed to broaden our understanding of the physiopathological role of these variants.

Previous reports have shown male sex to be an MEFV-independent modifying factor for FMF. The risk of renal amyloidosis was four times higher in males than in females, particularly in those patients who did not carry the M694V homozygous phenotype [Cazeneuve et al., 2000; Gershoni-Baruch et al., 2003]. Additionally, clinical manifestations of FCAS/MWS disease in an Indian kindred were milder in females than in males, and no affected female was known to have developed amyloidosis [McDermott et al., 2000]. Although the kindred we describe here is a small one, it is interesting that the male patient's clinical symptoms were far milder than those of the females, who, in addition, developed amyloidosis at the age of ∼50. Further studies are needed to understand the physiology by which the patient's sex can modify the risk of amyloidosis in FMF patients.

Finally, in order to investigate the presence of the amyloidosis-associated SAA1 α/α genotype [Cazeneuve et al., 2000] in the two patients who developed renal amyloidosis (no. 1 and 2) and to assess the genetic risk of amyloid deposition of the younger carriers (no. 4 and 5), the MEFV-independent modifier factor SAA1 gene polymorphism was analyzed. None of the patients were carriers of the amyloidosis-associated SAA1 α/α genotype, which suggests that renal amyloidosis in these patients could have been caused by the severe effect of the H478Y mutation itself.

Taken together, both the clinical features of the present kindred and the genetic data raise the question of whether the periodic autoinflammatory disorder seen in these patients can actually be considered a different MEFV-associated periodic inflammatory disorder, which would be dominantly inherited. In this connection, there are some examples of allelic diseases among the same group of HPF syndromes. Thus, mutations in the CIAS1/PYPAF1/NALP3 gene result in the phenotypically similar, yet distinct, periodic fever disorders of FCU/FCAS and MWS [Hoffman et al., 2001]. Furthermore, the R260W mutation was identified in two families with MWS and in two families with FCAS of different ethnic origins [Dode et al., 2002]. Furthermore, the V200M variant, was reported in a family with FCU/FCAS [Hoffman et al., 2001] and in an unrelated MWS kindred [Aganna et al., 2002], demonstrating that a single CIAS1/PYPAF1/NALP3 mutation may cause both syndromes. The phenotypical variation between these two conditions with the same apparent genetic basis may reflect subtle differences in the effects of different mutations or may be the result of differing genetic backgrounds or environmental factors [Hoffman et al., 2001]. Interestingly, the D303N mutation may result in MWS and also in a third CIAS1/PYPAF1/NALP3-associated disorder named CINCA/NOMID (Chronic Infantile Neurologic Cutaneous and Articular syndrome/neonatal onset multisystem inflammatory disease), an original entity defined by neonatal onset and a unique cluster of manifestations, including chronic polymorphonuclear cell meningitis, as well as the severe and characteristic joint manifestations, also caused by mutations in the CIAS1/PYPAF1/NALP3 gene [Feldmann et al., 2002].

Colchicine administration is the main therapy for FMF patients. In most cases it is able to control periodic attacks and prevent amyloid deposition, although it is relatively less effective in controlling joint involvement [Ben-Chetrit and Levy, 1998]. In view of the colchicine resistance in the present kindred and the severity of the disease associated with AA amyloidosis, biological therapy against TNFα was proposed, based on the important role of this cytokine in the pathogenesis of FMF and in the development of AA amyloidosis. Biological therapy blocks TNFα activity through a monoclonal antibody against TNFα (infliximab) or with the TNFα soluble receptor (etanercept) and has shown high efficacy in the treatment of rheumatoid arthritis, Crohn's disease, and spondyloarthropathy [Criscione and St-Clair, 2002]. Although the number of patients in the present study is limited and the follow-up short, the response to infliximab therapy in this kindred was clinically impressive, especially in the two young females. Both had a very severe form of HPF syndrome; infliximab treatment dramatically improved their clinical condition, completely blocked the periodic attacks, and even normalized the CRP in one patient (no. 4). Some reports have demonstrated the high efficacy of biological therapy in selective and severe cases, but experience with this therapy in amyloid diseases as well as in HPF syndromes is still limited [Drewe et al., 2000; Hull et al., 2002]. Thus, though the two younger patients currently lead normal lives, longer follow-up is necessary to confirm the benefits of infliximab in this family, especially as far as the prevention of AA amyloidosis is concerned.

Our results show that the H478Y MEFV mutation is a new variant associated with a severe autosomal-dominant periodic autoinflammatory disorder with AA amyloidosis, suggesting that this residue may play an important role in pyrin/marenostrin function. There are three main conclusions. First, the H478Y variant is a dominant-pathological MEFV variant. Second, full-length MEFV gene sequencing is needed to obtain adequate diagnosis of patients with clinical suspicion of a HPF syndrome, particularly patients from non-ancestral populations, in which private mutations are more frequent. Furthermore, we agree with many authors that the genetic diagnosis of HPF syndromes should be introduced in future revisions of the diagnostic criteria [Cazeneuve et al., 1999]. Third, mutations in the MEFV gene may also be responsible for an autosomal-dominant periodic inflammatory disorder accompanied by renal AA amyloidosis. Finally, administration of TNF biological therapy may be an alternative treatment in patients with this aggressive phenotype and colchicine unresponsiveness.


The authors are particularly grateful to all family members who agreed to participate in the study.