- Email: [email protected]
Two novel mutations R653H and E230K in the Mediterranean fever gene associated with disease
Mutation Research 479 (2001) 235–239
Short communication
Two novel mutations R653H and E230K in the Mediterranean fever gene associated with disease Christian Timmann∗ , Birgit Muntau, Kathrin Kuhne, Annette Gelhaus, Rolf D. Horstmann Department of Molecular Medicine, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359, Hamburg, Germany Received 9 March 2001; received in revised form 6 May 2001; accepted 1 June 2001
Abstract Familial Mediterranean fever (FMF) is an autosomal recessive disorder caused by mutations in the Mediterranean fever gene (MEFV). We describe two novel missense mutations in MEFV, R653H and E230K. Both were found in compound heterozygosity with the mutation M694V in single Turkish patients with clinical syndromes characteristic for FMF. DNA sequencing and PCR–RFLP typing of the families confirmed the mutations and verified recessive modes of inheritance. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Familial Mediterranean fever; FMF; Marenostrin; Pyrin; MEFV; Mutation
1. Introduction Familial Mediterranean fever (FMF; reviewed in [1]; OMIM, #249100 Mediterranean fever, familial) is characterized by self-limited attacks of fever, serositis, erysipelas-like skin disease, and the risk of developing amyloidosis. The disorder is predominantly found among individuals of non-Ashkenazi Jewish, Arab, Armenian, and Turkish origin. In the vast majority of cases, the inheritance pattern of the disease is consistent with an autosomal recessive model. A few cases with pseudodominant and dominant modes of inheritance have been reported [2]. Missense mutations, one nonsense mutation, and deletions of the ∗ Corresponding author. Tel.: +49-40-42818-516; fax: +49-40-42818-512. E-mail address: [email protected] (C. Timmann).
Mediterranean fever gene (MEFV) [3,4] were identified to cause FMF. The gene consists of 10 exons. One mutation was found in exon 1 (L110P [5]), three mutations were found in exon 2 (E148Q, E167D, T267I [6]), two in exon 3 (P369S [7], R408Q [8]), one in exon 5 (F479L [6]), and 14 in exon 10 (M694V, V726A [3], M680I 2040G>C, M694I [4], T681I, M694DEL [9], I692DEL, K695R, A744S, R761H [6], M680I 2040G>A [7], S675N, M680L [10], Y688X [11]), whereby M694V strongly predominates among Turks [12,13] and most other affected populations [7]. Recently, the prevalence of seven of the known MEFV mutations were determined by PCR–RFLP or allele-specific PCR among Turkish FMF patients. In 16% of the cases, none of the known mutations was found, and 24% of the patients were heterozygous for a single mutation [14]. It may be concluded that as yet unrecognized mutations are likely to exist [9].
0027-5107/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 ( 0 1 ) 0 0 2 2 1 - 4
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C. Timmann et al. / Mutation Research 479 (2001) 235–239
2. Materials and methods 2.1. Subjects Blood samples were obtained for diagnostic purposes from individuals with FMF symptoms. For clinical definitions of FMF, the Tel-Hashomer criteria were applied [15]. From the two families described here, informed consent was obtained for the documentation of their medical histories and for MEFV typing. 2.2. PCR amplification and sequencing Genomic DNA was isolated from blood using standard protocols. MEFV exons and the flanking intronic sequences were PCR amplified from genomic DNA using primers and conditions as compiled in Table 1. Standard PCR reactions were carried out in a final volume of 50 l 10 mM Tris–HCl, pH 9.0, containing 1.5 mM MgCl2 , 50 mM KCl, 200 M of each dNTP, 12.5 pmol of each primer, approximately 20 ng of genomic DNA, and 1.5 U Taq polymerase. Cycling
parameters included an initial denaturation step of 5 min at 94◦ C, 35 cycles consisting of 30 s at 94◦ C, 60 s at the annealing temperature, 60 s at 72◦ C, and a final extension step of 10 min at 72◦ C. Amplicons of the expected lengths were obtained as verified by electrophoresis in a 1.5% agarose gel. PCR products were purified using standard methods and were sequenced bidirectionally by automated DNA-sequencing. For allele discrimination in heterozygote individuals new amplicons were produced, cloned and sequenced using standard procedures. The coding sequence of the wild-type MEFV was obtained from the NCBI, Gene Bank accession number AF111163 (http://www.ncbi.nlm.nih.gov/). The novel mutations were deposited at the NCBI, Gene Bank accession number AF301150 for E230K and AF30115 for R653H. 2.3. PCR–RFLP Exon 2 was amplified as described above. Exon 10 was amplified partially. A 117 bp fragment was
Table 1 Primers and PCR conditions used Exon name
Exon sequence
Annealing temperature (◦ C)
Product size (bp)
Primer position (nt)a
Exon1F Exon1R Exon2F Exon2R Exon3Fd Exon3Rd Exon4Fd Exon4Rd Exon5Fd Exon5Rd Exon6Fd Exon6Rd Exon7&8F Exon7&8R Exon9F Exon9R Exon10aFd Exon10bRd Exon10R653HF Exon10R653HR
5 -TCCTACCAGAAGCCAGACAG 5 -TTCCTGAACTAAAGTCATCT 5 -GCATCTGGTTGTCCTTCCAGAATATTCC 5 -CTTTCTCTGCAGCCGATATAAAGTAGG 5 -GAACTCGCACATCTCAGGC 5 -AAGGCCCAGTGTGTCCAAGTGC 5 -TTGGCACCAGCTAAAGATGGC 5 -TCTCCCTCTACAGGGATGAGC 5 -TATCGCCTCCTGCTCTGGAATC 5 -CACTGTGGGTCACCAAGACCAAG 5 -TCCAGGAGCCCAGAAGTAGAG 5 -TTCTCCCTATCAAATCCAGAG 5 -TTGAGGACCAGCATTTAGAC 5 -GGCCAGCACACACCATTACC 5 -AAAGGTAGGAGAAAGGTCAT 5 -GGAAACAGGGACAGGGTAGT 5 -CCAGAAGAACTACCCTGTCCC 5 -TCCTCCTCTGAAATCCATGG 5 -CTCCGAGTTTCCTCTCTGGCGGCC 5 -GCGACAGAGTCATGTTCCCTT
57b
471
45c
696
60b
515
60b
331
60b
525
60b
277
55e
531
55b
374
60b
887
60b
117
963–982 1414–1433 2787–2814 3456–3482 7706–7724 8199–8220 8526–8546 8836–8856 10197–10218 10699–10721 10961–10981 11217–11237 12838–12857 13349–13368 13452–13471 13806–13825 13798–13818 14665–14684 14038–14061 14134–14154
a
According to genomic sequence (accession no. AF111163). Standard PCR conditions, see Section 2. c Reaction buffer contains 0.75 mM MgCl and in addition 5% DMSO. 2 d Described in [3]. e Reaction buffer contains 1.00 mM MgCl . 2 b
C. Timmann et al. / Mutation Research 479 (2001) 235–239
237
obtained using as primers Exon10R653HF, which introduces a restriction site specific for the wild-type allele, and Exon10R653HR (Table 1). The amplicons were analyzed using Taq I (MBI Fermentas) for mutation E230K in exon 2, and Eag I (New England BioLabs) for mutation R653H in exon 10. Approximately 100 ng of the PCR products were digested for 2 h in a volume of 10 l using buffers and conditions as recommended by the manufacturers. The digestion products were analyzed by agarose gel electrophoresis.
3. Results In the course of screening patients with clinical evidence for FMF, two individuals were found to be affected by novel missense mutations of MEFV. Patient 1 presented at the age of 3 years with a 1-year history of recurrent attacks of fever and peritonitis lasting 1 day. No invasive diagnostic procedures were applied to detect amyloidosis. PCR amplification and sequencing of exon 10 of MEFV showed two mutations, first, a nucleotide exchange 1958G>A causing an arginine-to-histidine exchange in codon 653 (R653H), and, second, a 2080A>G replacement resulting in M694V. Cloning and sequencing of a second amplification product revealed that the two mutations were located on separate alleles, indicating that the patient was compound heterozygous. Analysis of the complete MEFV coding sequence showed no further mutations affecting the protein structure. Enrollment of the first-degree relatives showed that none of them had any signs of FMF and that both parents were of Turkish origin. Amplification and sequencing of exon 10 revealed that the mother and the brother were carriers of R653H, whereas the father carried M694V. The segregation pattern of R653H was confirmed by PCR–RFLP of a 117 bp fragment of exon 10 using a forward primer modified to introduce a restriction site specific for the wild-type sequence (Fig. 1). Patient 2 was 9 years of age when she presented with a history of 5 years of febrile episodes with peritonitis lasting no longer than 3 days and occasionally accompanied by oligoarthritis and an erysipelas-like erythema. Again, no invasive diagnostic procedures had been applied to detect amyloidosis. Amplifications and sequencing of exon 10, exon 2, and subsequently all other exons of MEFV revealed two
Fig. 1. PCR–RFLP of novel MEFV mutations R653H and E230K analyzed by agarose gel electrophoresis. Left: family with the segregation of R653H. A restriction site specific for the wild-type sequence was introduced by primer modification. In the wild type, therefore, a 117 bp amplicon (A) of exon 10 was cleaved by Eag I into fragments of 22 bp (not visible) and 95 bp whereas the amplicon carrying the R653H allele remained uncleaved. Patient 1 (1), the mother (M) and the brother (B) were heterozygous for R653H and showed both the cleaved and uncleaved amplicons, whereas the father (F), with respect to R653H, was homozygous wild-type and showed the cleaved amplicon only. Right: family with the segregation of E230K. The E230K mutation abolishes one of two restriction sites for Taq I in exon 2, thereby changing the cleavage pattern of the 696 bp amplicon (A) from 204/226/266 to 204/492 bp. Patient 2 (2) and the father (F) were heterozygous for E230K and therefore showed a combination of the two restriction patterns. Regarding E230K, the mother (M) was homozygous wild type.
mutations. Exon 2 contained a 688G>A mutation, causing a glutamic acid-to-lysine exchange in codon position 230 (E230K). The second mutation, as in patient 1, was 2080A>G causing M694V in exon 10. The results were confirmed by re-amplification, cloning, and sequencing of exons 2 and 10. Both parents were of Turkish origin, and both were free of FMF symptoms. Amplification and sequencing of exons 2 and 10 showed each of them to be heterozygous for one of the mutations of the child indicating that the child is compound heterozygous. Again, the segregation pattern of the novel allele was confirmed by PCR–RFLP analysis (Fig. 1).
4. Discussion Two novel missense mutations were identified in MEFV. They cause amino acid replacements of glutamic acid by lysine in position 230 and of arginine
238
C. Timmann et al. / Mutation Research 479 (2001) 235–239
by histidine in position 653. Given the limited knowledge about the structure/function relationship of the MEFV protein, the functional influences of the two amino acid exchanges are difficult to predict. What one can state is that E230K causes a substantial change from a negatively to a positively charged side chain. In the case of R653H, the positively charged arginine residue is replaced by histidine, which is positively charged in an acidic microenvironment only. Of greater interest is the conclusion that both exchanges are very likely to cause FMF. This is supported by the following findings: (i) They were found in patients with clinical syndromes characteristic for FMF; (ii) their disease associations within families followed a recessive mode of inheritance, which is the established one for FMF [1]; (iii) they were found in compound heterozygosity with M694V, one of the best characterized mutations known to cause FMF in a recessive manner; (iv) no other mutations were found in the MEFV genes of the two affected individuals; and (v) the novel mutations were found in exon 2 and exon 10 of MEFV, where most of the previously identified FMF-causing mutations are located. Thus, we propose to add E230K and R653H to the list of MEFV mutations which may cause FMF. In order to avoid confusion with functionally irrelevant variants of MEFV such as R202Q [6] and possibly E148Q [16], the relevance of the novel mutations should be confirmed by studying the associations with FMF in large patient groups and by comparing the allele frequencies in FMF patients and controls. Routine diagnostics of FMF as well as large-scale screenings frequently rely on methods such as PCR–RFLP and amplification–refractory mutation screening, both of which are limited in that they detect known mutations only. In the future, such studies may include E230K and R653H by applying the PCR–RFLP assays presented here.
References [1] E. Ben-Chetrit, M. Levy, Familial Mediterranean fever, Lancet 351 (1998) 659–664. [2] D.R. Booth, J.D. Gillmore, H.J. Lachmann, S.E. Booth, A. Bybee, M. Soyturk, S. Akar, M.B. Pepys, M. Tunca, P.N.
[3]
[4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
Hawkins, The genetic basis of autosomal dominant familial Mediterranean fever, Q.J.M. 93 (2000) 217–221. The International FMF Consortium, Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever, Cell 90 (1997) 797–807. The French FMF Consortium, A candidate gene for familial Mediterranean fever, Nat. Genet. 17 (1997) 25–31. C. Domingo, I. Touitou, A. Bayou, S. Ozen, C. Notarnicola, M. Dewalle, J. Demaille, R. Buades, C. Sayadat, M. Levy, E. Ben-Chetrit, Familial Mediterranean fever in the Chuetas of Mallorca: a question of Jewish origin or genetic heterogeneity, Eur. J. Hum. Genet. 8 (2000) 242–246. A. Bernot, C. da Silva, J.L. Petit, C. Cruaud, C. Caloustian, V. Castet, M. Ahmed-Arab, C. Dross, M. Dupont, D. Cattan, N. Smaoui, C. Dode, C. Pecheux, B. Nedelec, J. Medaxian, M. Rozenbaum, I. Rosner, M. Delpech, G. Grateau, J. Demaille, J. Weissenbach, I. Touitou, Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever (FMF), Hum. Mol. Genet. 7 (1998) 1317–1325. I. Aksentijevich, Y. Torosyan, J. Samuels, M. Centola, E. Pras, J.J. Chae, C. Oddoux, G. Wood, M.P. Azzaro, G. Palumbo, R. Giustolisi, M. Pras, H. Ostrer, D.L. Kastner, Mutation and haplotype studies of familial Mediterranean fever reveal new ancestral relationships and evidence for a high carrier frequency with reduced penetrance in the Ashkenazi Jewish population, Am. J. Hum. Genet. 64 (1999) 949–962. C. Cazeneuve, T. Sarkisian, C. Pecheux, M. Dervichian, B. Nedelec, P. Reinert, A. Ayvazyan, J.C. Kouyoumdjian, H. Ajrapetyan, M. Delpech, M. Goossens, C. Dode, G. Grateau, S. Amselem, 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. 65 (1999) 88–97. D.R. Booth, J.D. Gillmore, S.E. Booth, M.B. Pepys, P.N. Hawkins, Pyrin/marenostrin mutations in familial Mediterranean fever, Q.J.M. 91 (1998) 603–606. C. Dode, C. Pecheux, C. Cazeneuve, D. Cattan, M. Dervichian, M. Goossens, M. Delpech, S. Amselem, G. Grateau, Mutations in the MEFV gene in a large series of patients with a clinical diagnosis of familial Mediterranean fever, Am. J. Med. Genet. 92 (2000) 241–246. C. Notarnicola, R. Manna, J.M. Rey, I. Touitou, Y688X, the first nonsense mutation in familial Mediterranean fever (FMF), Hum. Mutat. 17 (2001) 79. X. Chen, N. Fischel-Ghodsian, A. Cercek, M. Hamon, G. Ogur, R. Lotan, Y. Danon, M. Shohat, Assessment of pyrin gene mutations in Turks with familial Mediterranean fever (FMF), Hum. Mutat. 11 (1998) 456–460. F. Yalcinkaya, N. Cakar, M. Misirlioglu, N. Tumer, N. Akar, M. Tekin, H. Tastan, H. Kocak, N. Ozkaya, A.H. Elhan, Genotype-phenotype correlation in a large group of Turkish patients with familial Mediterranean fever: evidence for mutation-independent amyloidosis, Rheumatology 39 (2000) 67–72.
C. Timmann et al. / Mutation Research 479 (2001) 235–239 [14] N. Akar, M. Misiroglu, F. Yalcinkaya, E. Akar, N. Cakar, N. Tumer, M. Akcakus, H. Tastan, Y. Matzner, MEFV mutations in Turkish patients suffering from familial Mediterranean fever, Hum. Mutat. 15 (2000) 118–119. [15] A. Livneh, P. Langevitz, D. Zemer, N. Zaks, S. Kees, T. Lidar, A. Migdal, S. Padeh, M. Pras, Criteria for the diagnosis
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of familial Mediterranean fever, Arthritis Rheum. 40 (1997) 1879–1885. [16] E. Ben-Chetrit, I. Lerer, E. Malamud, C. Domingo, D. Abeliovich, The E148Q mutation in the MEFV gene: is it a disease-causing mutation or a sequence variant? Hum. Mutat. 15 (2000) 385–386.
Short communication
Two novel mutations R653H and E230K in the Mediterranean fever gene associated with disease Christian Timmann∗ , Birgit Muntau, Kathrin Kuhne, Annette Gelhaus, Rolf D. Horstmann Department of Molecular Medicine, Bernhard Nocht Institute for Tropical Medicine, Bernhard-Nocht-Strasse 74, D-20359, Hamburg, Germany Received 9 March 2001; received in revised form 6 May 2001; accepted 1 June 2001
Abstract Familial Mediterranean fever (FMF) is an autosomal recessive disorder caused by mutations in the Mediterranean fever gene (MEFV). We describe two novel missense mutations in MEFV, R653H and E230K. Both were found in compound heterozygosity with the mutation M694V in single Turkish patients with clinical syndromes characteristic for FMF. DNA sequencing and PCR–RFLP typing of the families confirmed the mutations and verified recessive modes of inheritance. © 2001 Elsevier Science B.V. All rights reserved. Keywords: Familial Mediterranean fever; FMF; Marenostrin; Pyrin; MEFV; Mutation
1. Introduction Familial Mediterranean fever (FMF; reviewed in [1]; OMIM, #249100 Mediterranean fever, familial) is characterized by self-limited attacks of fever, serositis, erysipelas-like skin disease, and the risk of developing amyloidosis. The disorder is predominantly found among individuals of non-Ashkenazi Jewish, Arab, Armenian, and Turkish origin. In the vast majority of cases, the inheritance pattern of the disease is consistent with an autosomal recessive model. A few cases with pseudodominant and dominant modes of inheritance have been reported [2]. Missense mutations, one nonsense mutation, and deletions of the ∗ Corresponding author. Tel.: +49-40-42818-516; fax: +49-40-42818-512. E-mail address: [email protected] (C. Timmann).
Mediterranean fever gene (MEFV) [3,4] were identified to cause FMF. The gene consists of 10 exons. One mutation was found in exon 1 (L110P [5]), three mutations were found in exon 2 (E148Q, E167D, T267I [6]), two in exon 3 (P369S [7], R408Q [8]), one in exon 5 (F479L [6]), and 14 in exon 10 (M694V, V726A [3], M680I 2040G>C, M694I [4], T681I, M694DEL [9], I692DEL, K695R, A744S, R761H [6], M680I 2040G>A [7], S675N, M680L [10], Y688X [11]), whereby M694V strongly predominates among Turks [12,13] and most other affected populations [7]. Recently, the prevalence of seven of the known MEFV mutations were determined by PCR–RFLP or allele-specific PCR among Turkish FMF patients. In 16% of the cases, none of the known mutations was found, and 24% of the patients were heterozygous for a single mutation [14]. It may be concluded that as yet unrecognized mutations are likely to exist [9].
0027-5107/01/$ – see front matter © 2001 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 ( 0 1 ) 0 0 2 2 1 - 4
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2. Materials and methods 2.1. Subjects Blood samples were obtained for diagnostic purposes from individuals with FMF symptoms. For clinical definitions of FMF, the Tel-Hashomer criteria were applied [15]. From the two families described here, informed consent was obtained for the documentation of their medical histories and for MEFV typing. 2.2. PCR amplification and sequencing Genomic DNA was isolated from blood using standard protocols. MEFV exons and the flanking intronic sequences were PCR amplified from genomic DNA using primers and conditions as compiled in Table 1. Standard PCR reactions were carried out in a final volume of 50 l 10 mM Tris–HCl, pH 9.0, containing 1.5 mM MgCl2 , 50 mM KCl, 200 M of each dNTP, 12.5 pmol of each primer, approximately 20 ng of genomic DNA, and 1.5 U Taq polymerase. Cycling
parameters included an initial denaturation step of 5 min at 94◦ C, 35 cycles consisting of 30 s at 94◦ C, 60 s at the annealing temperature, 60 s at 72◦ C, and a final extension step of 10 min at 72◦ C. Amplicons of the expected lengths were obtained as verified by electrophoresis in a 1.5% agarose gel. PCR products were purified using standard methods and were sequenced bidirectionally by automated DNA-sequencing. For allele discrimination in heterozygote individuals new amplicons were produced, cloned and sequenced using standard procedures. The coding sequence of the wild-type MEFV was obtained from the NCBI, Gene Bank accession number AF111163 (http://www.ncbi.nlm.nih.gov/). The novel mutations were deposited at the NCBI, Gene Bank accession number AF301150 for E230K and AF30115 for R653H. 2.3. PCR–RFLP Exon 2 was amplified as described above. Exon 10 was amplified partially. A 117 bp fragment was
Table 1 Primers and PCR conditions used Exon name
Exon sequence
Annealing temperature (◦ C)
Product size (bp)
Primer position (nt)a
Exon1F Exon1R Exon2F Exon2R Exon3Fd Exon3Rd Exon4Fd Exon4Rd Exon5Fd Exon5Rd Exon6Fd Exon6Rd Exon7&8F Exon7&8R Exon9F Exon9R Exon10aFd Exon10bRd Exon10R653HF Exon10R653HR
5 -TCCTACCAGAAGCCAGACAG 5 -TTCCTGAACTAAAGTCATCT 5 -GCATCTGGTTGTCCTTCCAGAATATTCC 5 -CTTTCTCTGCAGCCGATATAAAGTAGG 5 -GAACTCGCACATCTCAGGC 5 -AAGGCCCAGTGTGTCCAAGTGC 5 -TTGGCACCAGCTAAAGATGGC 5 -TCTCCCTCTACAGGGATGAGC 5 -TATCGCCTCCTGCTCTGGAATC 5 -CACTGTGGGTCACCAAGACCAAG 5 -TCCAGGAGCCCAGAAGTAGAG 5 -TTCTCCCTATCAAATCCAGAG 5 -TTGAGGACCAGCATTTAGAC 5 -GGCCAGCACACACCATTACC 5 -AAAGGTAGGAGAAAGGTCAT 5 -GGAAACAGGGACAGGGTAGT 5 -CCAGAAGAACTACCCTGTCCC 5 -TCCTCCTCTGAAATCCATGG 5 -CTCCGAGTTTCCTCTCTGGCGGCC 5 -GCGACAGAGTCATGTTCCCTT
57b
471
45c
696
60b
515
60b
331
60b
525
60b
277
55e
531
55b
374
60b
887
60b
117
963–982 1414–1433 2787–2814 3456–3482 7706–7724 8199–8220 8526–8546 8836–8856 10197–10218 10699–10721 10961–10981 11217–11237 12838–12857 13349–13368 13452–13471 13806–13825 13798–13818 14665–14684 14038–14061 14134–14154
a
According to genomic sequence (accession no. AF111163). Standard PCR conditions, see Section 2. c Reaction buffer contains 0.75 mM MgCl and in addition 5% DMSO. 2 d Described in [3]. e Reaction buffer contains 1.00 mM MgCl . 2 b
C. Timmann et al. / Mutation Research 479 (2001) 235–239
237
obtained using as primers Exon10R653HF, which introduces a restriction site specific for the wild-type allele, and Exon10R653HR (Table 1). The amplicons were analyzed using Taq I (MBI Fermentas) for mutation E230K in exon 2, and Eag I (New England BioLabs) for mutation R653H in exon 10. Approximately 100 ng of the PCR products were digested for 2 h in a volume of 10 l using buffers and conditions as recommended by the manufacturers. The digestion products were analyzed by agarose gel electrophoresis.
3. Results In the course of screening patients with clinical evidence for FMF, two individuals were found to be affected by novel missense mutations of MEFV. Patient 1 presented at the age of 3 years with a 1-year history of recurrent attacks of fever and peritonitis lasting 1 day. No invasive diagnostic procedures were applied to detect amyloidosis. PCR amplification and sequencing of exon 10 of MEFV showed two mutations, first, a nucleotide exchange 1958G>A causing an arginine-to-histidine exchange in codon 653 (R653H), and, second, a 2080A>G replacement resulting in M694V. Cloning and sequencing of a second amplification product revealed that the two mutations were located on separate alleles, indicating that the patient was compound heterozygous. Analysis of the complete MEFV coding sequence showed no further mutations affecting the protein structure. Enrollment of the first-degree relatives showed that none of them had any signs of FMF and that both parents were of Turkish origin. Amplification and sequencing of exon 10 revealed that the mother and the brother were carriers of R653H, whereas the father carried M694V. The segregation pattern of R653H was confirmed by PCR–RFLP of a 117 bp fragment of exon 10 using a forward primer modified to introduce a restriction site specific for the wild-type sequence (Fig. 1). Patient 2 was 9 years of age when she presented with a history of 5 years of febrile episodes with peritonitis lasting no longer than 3 days and occasionally accompanied by oligoarthritis and an erysipelas-like erythema. Again, no invasive diagnostic procedures had been applied to detect amyloidosis. Amplifications and sequencing of exon 10, exon 2, and subsequently all other exons of MEFV revealed two
Fig. 1. PCR–RFLP of novel MEFV mutations R653H and E230K analyzed by agarose gel electrophoresis. Left: family with the segregation of R653H. A restriction site specific for the wild-type sequence was introduced by primer modification. In the wild type, therefore, a 117 bp amplicon (A) of exon 10 was cleaved by Eag I into fragments of 22 bp (not visible) and 95 bp whereas the amplicon carrying the R653H allele remained uncleaved. Patient 1 (1), the mother (M) and the brother (B) were heterozygous for R653H and showed both the cleaved and uncleaved amplicons, whereas the father (F), with respect to R653H, was homozygous wild-type and showed the cleaved amplicon only. Right: family with the segregation of E230K. The E230K mutation abolishes one of two restriction sites for Taq I in exon 2, thereby changing the cleavage pattern of the 696 bp amplicon (A) from 204/226/266 to 204/492 bp. Patient 2 (2) and the father (F) were heterozygous for E230K and therefore showed a combination of the two restriction patterns. Regarding E230K, the mother (M) was homozygous wild type.
mutations. Exon 2 contained a 688G>A mutation, causing a glutamic acid-to-lysine exchange in codon position 230 (E230K). The second mutation, as in patient 1, was 2080A>G causing M694V in exon 10. The results were confirmed by re-amplification, cloning, and sequencing of exons 2 and 10. Both parents were of Turkish origin, and both were free of FMF symptoms. Amplification and sequencing of exons 2 and 10 showed each of them to be heterozygous for one of the mutations of the child indicating that the child is compound heterozygous. Again, the segregation pattern of the novel allele was confirmed by PCR–RFLP analysis (Fig. 1).
4. Discussion Two novel missense mutations were identified in MEFV. They cause amino acid replacements of glutamic acid by lysine in position 230 and of arginine
238
C. Timmann et al. / Mutation Research 479 (2001) 235–239
by histidine in position 653. Given the limited knowledge about the structure/function relationship of the MEFV protein, the functional influences of the two amino acid exchanges are difficult to predict. What one can state is that E230K causes a substantial change from a negatively to a positively charged side chain. In the case of R653H, the positively charged arginine residue is replaced by histidine, which is positively charged in an acidic microenvironment only. Of greater interest is the conclusion that both exchanges are very likely to cause FMF. This is supported by the following findings: (i) They were found in patients with clinical syndromes characteristic for FMF; (ii) their disease associations within families followed a recessive mode of inheritance, which is the established one for FMF [1]; (iii) they were found in compound heterozygosity with M694V, one of the best characterized mutations known to cause FMF in a recessive manner; (iv) no other mutations were found in the MEFV genes of the two affected individuals; and (v) the novel mutations were found in exon 2 and exon 10 of MEFV, where most of the previously identified FMF-causing mutations are located. Thus, we propose to add E230K and R653H to the list of MEFV mutations which may cause FMF. In order to avoid confusion with functionally irrelevant variants of MEFV such as R202Q [6] and possibly E148Q [16], the relevance of the novel mutations should be confirmed by studying the associations with FMF in large patient groups and by comparing the allele frequencies in FMF patients and controls. Routine diagnostics of FMF as well as large-scale screenings frequently rely on methods such as PCR–RFLP and amplification–refractory mutation screening, both of which are limited in that they detect known mutations only. In the future, such studies may include E230K and R653H by applying the PCR–RFLP assays presented here.
References [1] E. Ben-Chetrit, M. Levy, Familial Mediterranean fever, Lancet 351 (1998) 659–664. [2] D.R. Booth, J.D. Gillmore, H.J. Lachmann, S.E. Booth, A. Bybee, M. Soyturk, S. Akar, M.B. Pepys, M. Tunca, P.N.
[3]
[4] [5]
[6]
[7]
[8]
[9]
[10]
[11]
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