Mutational spectrum in the MEFV and TNFRSF1A genes in patients suffering from AA amyloidosis and recurrent inflammatory attacks
Catherine Dodé1,2,
Bouke PC Hazenberg3,
Christophe Pêcheux1,
Daniel Cattan4,
Bruno Moulin5,
Anne Barthélémy6,
Marie-Claire Gubler7,
Marc Delpech1,2 and
Gilles Grateau2,8,
1 Laboratoire de biochimie et génétique moléculaire, Hôpital Cochin, Assistance Publique-Hôpitaux de Paris (AP-HP), Paris,
2 Institut National de la Santé et de la Recherche Médicale (EMI 0005), ICGM, Université Paris V, Paris, France,
3 Department of Rheumatology, University Hospital, Groningen, The Netherlands,
4 Service d'hépato-gasto-entérologie, Centre hospitalier, Villeneuve-Saint-Georges,
5 Service de néphrologie, Hôpital civil et d'hémodialyse, Strasbourg,
6 Service de néphrologie pédiatrique, Hôpital Necker, AP-HP, Paris,
7 Institut National de la Santé et de la Recherche Médicale (Unité 423), Hôpital Necker, AP-HP, Paris and
8 Service de médecine interne, Hôtel-Dieu, Paris, France
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Abstract
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Background. Among hereditary fevers characterized by recurrent attacks of fever and organ localized inflammation, familial Mediterranean fever (FMF), and tumour necrosis factor receptor superfamily 1A (TNFRSF1A) receptor associated periodic syndrome (TRAPS) are diseases with identified genes that can be associated with renal amyloidosis of the AA type. In this study we have characterized FMF and TRAPS genotypes in 38 unrelated patients suffering from amyloidosis AA and recurrent inflammatory attacks.
Methods. Mutations of the MEFV and TNFRSF1A genes, responsible respectively for FMF and TRAPS, were searched for by amplifying, using polymerase chain reaction (PCR), genomic DNA, and direct sequencing.
Results. Twenty-seven patients (71%) carried mutations in MEFV (22 patients with two mutations, two patients with a single mutation) or TNFRSF1A genes (three patients). Patients with MEFV mutations belonged to the classical at-risk ethnic group for FMF: Sephardic Jews, Turks, Armenians, and Arabs from the Maghreb. The main genotype encountered was M694V/M694V (19/22), one Turkish patient was M680I/M680I, and two Arab patients from the Maghreb were M694I/M694I. We found three Caucasian patients with the C55S, C70Y, R92Q mutations in the TNFRSF1A gene.
Conclusions. In this series we observed that FMF is the main cause of AA amyloidosis in Sephardic Jews and Turks. MEFV and TNFRSF1A mutations were found in only 6 of 14 Arab patients from the Maghreb. We found three families (one Caucasian and two from Maghreb) with AA amyloidosis without MEFV or TNFRSF1A mutations, suggesting that other genetic cause(s) exist(s). The characterization of mutations in MEFV and TNFRSF1A is important for the therapeutic behaviour of AA amyloidosis associated with inherited recurrent fever.
Keywords: amyloidosis AA; familial Mediterranean fever; inherited recurrent fever; MEFV; tumour necrosis factor receptor superfamily 1A receptor associated periodic syndrome
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Introduction
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Renal amyloidosis of the AA type is the main complication of hereditary diseases such as familial Mediterranean fever (FMF) (MIM 249100) and more recently described disorders such as tumour necrosis factor receptor superfamily 1A (TNFRSF1A)-associated periodic syndrome (TRAPS) (MIM 142680) and MuckleWells syndrome (MWS) (MIM 191900) [1]. These diseases are disorders of the inflammatory pathway characterized by recurrent attacks of fever with visceral, synovial, or cutaneous inflammation. Although in FMF amyloidosis generally appears years after the beginning of inflammatory attacks (phenotype I), it may develop in some patients without or prior to other manifestations (phenotype II).
FMF is an autosomal recessive disorder. The gene involved in FMF (designated MEFV) has been identified by positional cloning [2,3]. FMF affects populations of the Mediterranean basin such as Sephardic Jews, Armenians, Turks, and Arabs from Maghreb. Diagnosis of FMF leads to the beginning of a daily and life-long administration of colchicine that is an efficient treatment of both the attacks and of amyloidosis [4].
MWS and TRAPS are transmitted on the autosomal-dominant mode and seem to affect mainly North-European populations. In the MuckleWells syndrome, urticaria and conjunctivitis are observed in addition to arthritis. Later in the course of the disease, nerve deafness occurs. The development of renal AA amyloidosis can occur after several years of inflammatory conditions [5,6]. No treatment has been proved to prevent the AA amyloidosis. Linkage analysis allowed localizing of the MWS gene at the 1q44 chromosomic region [7], since no gene has yet been assigned to this disease. A North Indian family with AA amyloidosis and clinical features overlapping those of MWS and TRAPS, linked to the 1q44 chromosomic region, has recently been reported [8], suggesting that MWS may not be restricted to North European populations.
TRAPS is characterized by longer inflammatory attacks, often more than 1 week, than in FMF, associated with myalgias and sometimes periorbital oedema and rash that progressed centrifugally to the limbs [9]. Mutations found so far are localized in the extracellular part of the receptor in the two N terminal cysteine-rich domains. Mutations affect residues that play crucial roles in the structure and the function of the receptor. It has been observed than during inflammatory crisis in patients suffering from TRAPS, the level of soluble TNFRSF1A (sTNFRSF1A) remained abnormally low and it has been assumed that the antagonistic role of the sTNFRSF1A is therefore dramatically reduced [9]. Galon et al. [10] estimated that 25% of TRAPS patients developed AA amyloidosis. Colchicine is inefficient in TRAPS, whereas steroids and etanercept seem to attenuate inflammatory attacks and could even lead to the regression of the nephrotic syndrome [1012].
In this study we investigated MEFV and TNFRSF1A gene mutations in 38 patients with AA amyloidosis associated with recurrent inflammatory attacks. The goals of this study were (i) to determine the spectrum of mutations involved in FMF and TRAPS associated with AA amyloidosis, (ii) to try to refine clinical features of both diseases. Finding mutations in patients is important for managing the disease, therapeutic orientation that is different between FMF and TRAPS, and for genetic counselling in the family.
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Subjects and methods
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Subjects
We investigated 38 unrelated patients in whom AA amyloidosis was associated with recurrent inflammatory attacks and no obvious cause of underlying inflammatory disorder usually associated with AA amyloidosis (such as rheumatoid arthritis, spondylarthropathy, or Crohn's disease). Among these patients, 33 belonged to populations at risk for FMF, living in France, with 14 Arabs from the Maghreb, nine Sephardic Jews, eight Turks, one Armenian, and one Ashkenazi Jew; five patients with AA amyloidosis were Caucasians. Clinical features including fever, abdominal pains, arthritis, and age at onset, were recorded prior to therapy on a standardized form. In all of these patients, MEFV and TNFRSF1A genotypes were done.
Methods
Histology
In all affected patients, tissue specimens with amyloid deposits were obtained. The diagnosis of amyloidosis was made on a positive Congo-red staining, with typical birefringence under polarized light. The nature of amyloid deposits was determined by immunoperoxidase staining using specific antisera against SAA protein (Calbiochem, La Jolla, CA, USA) or by Wright's permanganate method.
DNA extraction
Genomic DNA was isolated from the patient's peripheral-blood leukocytes using standard procedures [13].
Mutation analysis in MEFV gene (Gene bank accession no. Y14441)
Mutations presented in exon 10 between codons 663 and 771 including the four most frequent mutations, namely M680I, M694V, M694I, V726A and the mutation E148Q in exon 2 were searched for using the procedure previously described [14].
Mutation analysis in TNFRSF1A gene (Gene bank accession no. M75866)
Mutation search in the TNFRSF1A gene was realized in patients heterozygous or without MEFV mutations. Polymerase chain reaction (PCR) amplification followed by sequencing of the 10 exons of the TNFRSF1A gene, were performed as previously described [15]. The PCR products were directly sequenced with primers used in the PCR reaction.
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Results
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Mutation analysis in MEFV and TNFRSF1A genes was carried out in 38 patients suffering from renal AA amyloidosis. The results of the different genotypes and clinical data are presented in Table 1
. As was previously known MEFV mutations were found only in patients belonging to ethnic origins at risk of FMF such as Sephardic Jews, Turks, Armenians and Arabs from the Maghreb [1618]. In our series, 22/33 patients displayed a homozygous MEFV mutation. The M694V/M694V genotype found in 19/22 patients was the main one associated with AA amyloidosis. In this series, other genotypes were also found: the M680I/ M680I genotype was observed in a Turkish patient and the M694I/M694I in two Arab patients. In two of 33 patients, a single FMF mutation was found (M694V, M694I). Among patients with a single mutation or without MEFV mutation, we searched for mutations in the TNFRSF1A gene. PCR fragments corresponding to the complete coding region of the TNFRSF1A gene exons (exons 110) were amplified, followed by direct sequencing. Three different mutations were found in three unrelated patients. Two patients were Dutch and displayed the C70Y (patient GVM) [19] and the R92Q mutations (patient DD) [11]; a Belgian patient carried the C55S (patient JD) [20].
Clinical signs of the 38 patients are summarized in Table 1
. The sex ratio (M/F) was: 16/6 for FMF patients with two mutations, 2/1 for TRAPS patients, and 5/6 for patients without FMF or TRAPS mutations. For both diseases FMF and TRAPS, the age at onset was between 1 and 14 years and attacks were mainly characterized by fever associated with abdominal signs and/or arthritis.
The patient DD who carried the R92Q mutation (individual III-5 on Figure 1A
), developed AA amyloidosis and needed a renal transplant after several years of inflammatory attacks. Since renal transplantation, he has not suffered any attacks. The father of the propositus II-4 had renal insufficiency some years before his death. The sister III-2 of the propositus had periods of fever and abdominal complaints. Patient IV-1 had recurrent periodic fever and was suspected of having hyperimmunoglobulinaemia D and periodic fever syndrome (MIM 260920) because of elevated IgD. He died in an accident at the age of 25 and was not tested for TRAPS mutations. His brother IV-2 carried the R92Q mutation but at the time of writing he remains asymptomatic. The R92Q mutation abolished a NciI restriction site (data not shown). In order to rule out the possibility that this nucleotide substitution corresponds to a polymorphism, PCR amplification of the TNFRSF1A exon 4 followed by NciI digestion of the PCR product was performed on 156 alleles from North-European individuals. The NciI site was present in all control alleles, suggesting that the R92Q mutation was responsible of the disease in family A.

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Fig. 1. Some pedigrees of families with familial recurrent fevers and AA amyloidosis. Family A, TRAPS with R92Q mutation; families B, C, and D, without mutation in MEFV and TNFRSF1A genes. Patients are represented with solid symbols and asymptomatic subjects with grey symbols.
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In this series and in the Sephardic and Turkish population groups, FMF was the main cause of AA amyloidosis with, respectively, 8 of 9 and 8 of 8 homozygotic patients in Sephardic and Turkish populations. In the Maghrebian patients, AA amyloidosis was due to MEFV mutations in only 5 of 14 cases, suggesting another cause(s) for the disease. In at least two cases, this cause seemed to be due to an inherited disease. In a Tunisian family (Figure 1B
) and an Algerian family (Figure 1C
), parents of the propositus were first cousins, two patients in each of these families have been affected by AA amyloidosis and had no MEFV or TNFRSF1A mutations. Patient IV-2 (Figure 1B
) had suffered from inflammatory attacks with fever and arthritis since the age of 3 years with a good response to colchicine therapy. At the age of 5 years, both rectal and renal biopsies showed evidence of AA amyloidosis. At the age of 9 years, hepatic AA amyloidosis was diagnosed. His sister IV-1 had the same clinical evolution and died of renal insufficiency. The patient II-2 (Figure 1C
), originating from Algeria, presented the first clinical signs of FMF in the first months of life; he then received colchicine treatment. At the age of 11 years, a nephrotic syndrome due to AA amyloidosis was observed. His sister II-1 with a similar clinical history died of AA amyloidosis at the age of 12. The nephrotic syndrome was resistant to corticosteroid therapy.
The last family is Caucasian; at the age of 15 years, the patient II-2 (Figure 1D
) started recurrent episodes of fever and abdominal pain associated with diarrhoea. Rectal and renal biopsies showed AA amyloidosis. Her sister had died of renal insufficiency a few years previously.
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Discussion
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The M694V/M694V genotype was the main one encountered in FMF patients with AA amyloidosis as previously described [16,17,21]. In our series, two other genotypes were observed M680I/M680I and M694I/M694I. This indicated that the M694V/ M694V genotype was not the only one responsible for AA amyloidosis as it was previously reported in the Turkish [2224] and Armenian patients [21]. Phenotype II (patients 53801 and 54001, Table 1
) has been observed in two patients who started to have recurrent episodes of fever associated with abdominal pain after renal transplantation. The MEFV genotypes of patients 53801 and 54001 are respectively M694V/ M694V and M680I/M680I. Until now the only MEFV genotype found to be associated with phenotype II was M694V/M694V [17]. We present a description of another MEFV genotype, M680I/M680I, in association with phenotype II.
Two patients without FMF family background were heterozygous for MEFV mutations (M694V, M694I) without TNFRSF1A mutations. These genotypes could not explain the occurrence of renal AA amyloidosis unless a second mutation had not yet been identified, as we did not sequence the complete coding region of the MEFV gene. It has also been reported that patients with a single MEFV mutation could be suffering from FMF and develop AA amyloidosis, in families with a dominant inheritance trait [25].
TRAPS is due to mutations in the two first N terminal cysteine-rich domain (CRD1 and 2) of the extracellular part of TNFRSF1A. At this time, 16 mutations have been reported, nine in CRD1 (H22Y, C30S, C30R, C33Y, C33G, P46L, T50M, C52F, c19314G>A) and seven in CRD2 (C55S, C70Y, S86P, C88R, C88Y, R92P, R92Q) [9,11,15,19,20]. We report here three TRAPS mutations in patients suffering from AA amyloidosis, in CRD2: C55S, C70Y, and R92Q (Figure 2
).

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Fig. 2. Mutated amino acids in the CRD1 and CRD2 domains of TNFRSF1A involving in TRAPS. The extracellular bonds are shown as horizontal lines between cysteine residues in CRD1, the same structure is observed in CRD2. Vertical dashed lines represent the alignment of cysteine residues. Underlined mutations are those presented in this study. The mutation c19314G>A is not shown in this figure.
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The R92 residue is located in the loop 3 of the CRD2 domain in a region of hydrogen bonds important for the stabilization of the domain structure [26]. In position 92, Aganna et al. [27] identified another mutation R92P, which allowed us to hypothisize that this amino acid is subjected to a mutational hotspot as other residues: C30, C33 and C88 (Figure 2
). In a recent report [11], it has been postulated that non-cysteine mutations were not associated (except one case with the T50M mutation) with amyloidosis AA. Family A is the first familial TRAPS case with amyloidosis AA and a R92Q mutation.
Recently, modifying genetic factors for FMF have been found to modulate the expression of the disease in Armenian patients [28]. The SAA1
/
genotype was associated with a 7-fold increased risk for renal amyloidosis compared with other SAA1 genotypes, and the risk for male patients of developing renal amyloidosis was 4-fold higher than that for female patients. In our study we observed that the male: female ratio was 2.5 (16/6) for FMF patients. It will be interesting to assess the SAA1, SAA2, and APOE genotypes in FMF and in TRAPS patients affected by these diseases. It will be particularly interesting to assess relatives of TRAPS patients carrying TNFRSF1A mutation either without or with few clinical signs, in order to evaluate the risk of amyloidosis.
We have also observed that in three families (Figure 1B
D
) with several patients suffering from AA amyloidosis, no FMF or TRAPS mutation were detected. This observation implies that other gene(s) is (are) responsible for AA amyloidosis, such as for example MWS. Unfortunately, the three families were too small to check for a possible transmission with the gene(s) underlying MWS. In families 1B, 1C the parents of the propositus were first cousins, so the transmission of the disease seemed likely to be autosomal recessive rather than dominant as in MWS. The parents of the sporadic patient 22601 (Table 1
), who is also negative for FMF and TRAPS mutations, were also first cousins.
In most series of AA amyloidosis, the underlying disorder appears to be unknown or not well characterized in 15% of patients [29]. Genetic diseases, mainly with a sporadic presentation, could be responsible for some of these underlying inflammatory disorders. Among them, FMF is no more isolated and TRAPS should be recognized and diagnosed with the help of molecular genetics, since its treatment relies on specific drugs.
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Acknowledgments
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We would like to thank patients for their participation in our study. The authors wish to thank Drs Olivier Benveniste, Nezly-Lynda Bererhi, Axel Glunz, Luisa Lobato, Sytze Meijer, Jean-Philippe Méry, Jean-Charles Piette, and Aziz Sefiani for sending blood samples for mutational analysis. This work was supported by l'Association Française contre les Myopathies (AFM), le Programme Hospitalier de Recherche Clinique (1997), and the INSERM-AFM network Hereditary recurrent inflammatory disorders.
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Notes
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Correspondence and offprint requests to: Dr G. Grateau, Hôtel-Dieu, Service de médecine interne, 75004 Paris. Email: gilles.grateau@htd.ap-hop-paris.fr 
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Received for publication: 28. 8.01
Accepted in revised form: 5. 1.02