Familial Mediterranean fever gene and protection against asthma

Familial Mediterranean fever gene and protection against asthma Einat Rabinovitch, MD*; Dror Harats, MD†‡; Pnina Yaron, MA§; Tamar Luvish, BSc†; Merav Lidar, MD*; Ron Kedem, PhD*; Aviv Shaish, PhD†; Issahar Ben-Dov, MD‡§; and Avi Livneh, MD*‡

Background: Asthma is an inflammatory airway disease caused by interaction between susceptibility genes and diverse environmental factors. In Israel, asthma seems to be familial and more severe in patients of Iraqi Jewish descent. On the other hand, asthma is less frequent in individuals with familial Mediterranean fever, an autoinflammatory disease prevalent in the Iraqi Jewish community and linked to mutations in the familial Mediterranean fever gene, designated MEFV. Objectives: To explore a possible role for mutated MEFV in the reduced susceptibility to asthma and to determine its expression in Israeli subjects of Iraqi origins. Methods: Using a case-control approach, we studied the presence of the 3 most common MEFV mutations (M694V, V726A, and E148Q) in DNA samples from 75 patients with asthma and 45 asymptomatic first-degree relatives, all of Iraqi Jewish origin. The severity of asthma was evaluated using a published severity score. Results: Eleven patients with asthma and 14 of their relatives carried 1 or 2 mutations in the MEFV gene, a carrier rate significantly lower in patients with asthma than in their first-degree relatives and in ethnically matched healthy individuals (P ⬍ .03 and P ⬍ .003, respectively). Carriers of MEFV mutations had less severe disease, compared with noncarriers (P ⬍ .002). Conclusion: These findings suggest that MEFV mutations may have a protective effect in the pathogenesis of asthma. Ann Allergy Asthma Immunol. 2007;99:517–521.

INTRODUCTION Asthma is a common inflammatory airway disease that occurs in genetically susceptible individuals on exposure to insults that trigger airway inflammation, bronchial hyperresponsiveness, and airway remodeling.1 The disease is classified as a genetically complex disease, with linkage to several genomic regions in chromosomes 1, 2, 5, 7, 9, 11, 12, and 16.2– 6 Familial Mediterranean fever (FMF) is a genetic disease with autosomal recessive transmission, characterized by recurrent short bouts of serous inflammation with fever and a clinical picture of peritonitis, pleuritis, and/or synovitis.7 The MEditerranean FeVer (MEFV) gene, thought to cause FMF, has been cloned and found to encode a 781–amino acid protein, designated pyrin or marenostrin,8,9 with an uncertain function, yet thought to have an important role in pathways leading to inflammation and apoptosis.10 Several founder MEFV mutations have been identified, the most common of which are M694V, V726A, and E148Q.8,9,11

* Heller Institute of Medical Research, Sheba Medical Center, TelHashomer, Israel. † Lipids and Atherosclerosis Research Center, Sheba Medical Center, TelHashomer, Israel. ‡ Sackler Faculty of Medicine, Tel Aviv University, Tel-Aviv, Israel. § Pulmonary Institute, Sheba Medical Center, Tel-Hashomer, Israel. Authors have nothing to disclose. Received for publication January 9, 2007. Received in revised form May 22, 2007. Accepted for publication June 10, 2007.

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Identification of the pyrin protein was followed by revolutionary developments in the understanding of autoinflammatory diseases, leading to a definition of new families of proteins and inflammatory pathways, in which the pyrin domain (part of the pyrin protein and of the apoptosis-associated specklike protein containing a caspase recruitment domain, abbreviated as ASC, and of protein families designated CATRPILLER, and other families of proteins) serves as a major constituent with a role in homophilic proteinprotein interaction and a significance much beyond its role in pyrin protein of FMF.12–14 Nevertheless, a linkage between pyrin-associated proteins and asthma has not yet been studied. Danon et al15 found that the rate of asthma in 800,000 new military recruits of the Israeli defense forces is 3%. In this cohort, there were 870 patients with FMF, of whom only 1% had asthma in addition to FMF.15 A number of much smaller studies demonstrated a nonsignificant trend for lower prevalence of asthma in patients with FMF and in individuals heterozygous for an MEFV mutation.16,17 No explanation was offered for this apparently negative association between the diseases. However, with the better understanding of the central role of pyrin in inflammation, it was possible that mutated MEFV may protect individuals from the development of asthma. Familial Mediterranean fever is prevalent in Israel in nonAshkenazi (Sephardic) Jews, of whom the Iraqi community is noted for the highest frequency of MEFV mutation carriage (1:4).18 This population is also affected by a high rate of severe familial asthma and, therefore, is an excellent population

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Table 1. Distribution of DNA Samples From Patients and Unaffected Individuals in Families With Familial Asthma

tation of our institution approved the study. Informed consent was not required for testing the archived DNA samples.

No. of families contributing No. of subjects participating from an individual family 1 2 3 4 5 6 8 Total

Only subjects with asthma

Only subjects without asthma

Both subjects with and without asthma

15 1 2 2 0 0 0 20

5 1 0 1 0 0 0 7

0 5 3 4 3 2 2 19

Total

20 7 5 7 3 2 2 46

to explore the negative association between the 2 disease entities, thus creating the objective of the present study. METHODS Study Design In a case-control manner and a setting of patients receiving treatment in a hospital pulmonary clinic, we investigated the presence of common MEFV mutations in genomic DNA of Iraqi Jewish patients with asthma and their asymptomatic first-degree relatives and compared the severity of asthma in MEFV mutation carriers with that of noncarriers. Study and Control Groups In this study, we used clinical and demographic data and DNA samples, collected during 1996 from patients and their first-degree relatives, from families with familial asthma, for Asthmagene, a bank of DNA samples and data established for studies on the genetics of asthma. Families, eligible for the original database and sample collection, had to consist of at least 2 siblings with asthma and 1 unaffected first-degree family member. Blood samples for DNA extraction were obtained only from a fraction of patients and relatives. Seventy-five adults with asthma (study group) and 45 nonasthmatic (by their statement, physician evaluation, and pulmonary function test results) relatives of patients with asthma (control group) were included in the study. All patients and individuals of the control group were of Iraqi Jewish origin. None of the patients or control individuals had FMF. Asthma in the patients was expressed with typical manifestations and pulmonary function test results, consistent with published guidelines for the diagnosis of asthma.19 All patients of the study group had their conditions diagnosed and were followed up at the Pulmonary Institute of Sheba Medical Center. All current and past smokers and patients with cardiac disease and occupational lung diseases were excluded from the study. The severity of asthma was estimated using the National Asthma Education and Prevention Program severity scale.19 The Ethic Committee for Human Experimen-

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Detection of Common MEFV Mutations The 3 most common MEFV mutations in the Iraqi Jewish population (M694V, V726A, and E148Q) were determined. Mutation analyses were performed using polymerase chain reaction amplification of the DNA segment of interest and restriction enzyme digestion, as described previously.20 Statistical Analysis Statistical analysis was performed using the Fisher exact test for discrete and categorical variables and the t test for continuous variables. Because we assumed that asthma protection is conferred by MEFV mutations, the null hypothesis was that MEFV mutations are not less frequent in the study population and, therefore, studies looking for a reduced frequency of MEFV-mutated alleles in patients with asthma were 1-tailed. All the other studies were 2-tailed. P ⬍ .05 was considered statistically significant. RESULTS We determined the presence of 3 common MEFV mutations in 120 DNA samples from 75 patients with asthma and 45 nonasthmatic first-degree relatives. Each of the 46 participating families contributed 1 to 8 members, including patients, control individuals, or both (Table 1). No statistically significant differences between patients and family member controls were found with respect to their mean ⫾ SD age (43.4 ⫾ 21.1 vs 41.2 ⫾ 19.8 years; P ⫽ .60) and sex (males, 34 [45%] vs 23 [51%]; P ⬎ .50). The MEFV mutations found included V726A in 6 patients with asthma (7 in unaffected first-degree relatives), E148Q in 4 patients with asthma (8 in relatives), and M694V in 1 patient with asthma (3 in relatives). Of the healthy firstdegree relatives, 4 were compound heterozygotes: 1 carried V726A and E148Q mutations, 2 carried M694V and E148Q mutations, and 1 carried V726A and M694V mutations. These individuals had no febrile attacks or manifestations that could be attributed to FMF. Altogether, we found 29 mutations in 240 alleles with specific allelic distribution, which is similar to prior results found in the Iraqi Jewish population.20,23,24 The rate of mutation carriers is summarized in Table 2. Contrary to unaffected relatives and to the expected frequency in the same ethnic population, patients with asthma were featured by a significantly lower frequency of carriers. To account for the individuals bearing 2 mutations, we compared the frequencies of mutated alleles, computed for the number of chromosomes at risk in each group (patients vs unaffected relatives) and found the difference between the groups to be significant (11 of 150 alleles vs 18 of 90 alleles; P ⫽ .003). Focusing, however, only on the 19 of the 46 families that contributed to the study both patients with asthma and unaffected relatives, the results seem to be less unequivocal. In these 19 families, there were 44 patients with asthma (6

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Table 2. Rate of MEFV Mutation Carriers in the Patient and Control Groups No. of MEFV Frequency mutation P value of carriers carriers

Studied population

No. of subjects

Patients with asthma Unaffected family members Unrelated ethnically matched healthy control subjectsc

75 45

11 14a

0.14 0.32

NA ⬍.03b

101

34

0.30

⬍.003d

Abbreviations: MEFV, MEditerranean FeVer; NA, data not applicable. a In 4 patients, 2 mutations were found, making 18 total mutations. b Patients with asthma vs family members. c Determined in a separate study17 of the MEFV mutation rate in Iraqi Jewish healthy subjects, performed by the same laboratory and personnel. d Patients with asthma vs unrelated controls.

carriers, each with 1 mutated allele) and 32 unaffected individuals (9 carriers, with 11 mutated alleles). Although mutation carriage was almost twice as much in the first-degree unaffected relatives group (6 of 44 individuals vs 9 of 32 individuals), only allelic mutation analysis yielded a significant difference between this group and the asthma patient group (6 of 88 related chromosomes for patients with asthma vs 11 of 64 related chromosomes for unaffected relatives; P ⫽ .04). Table 3 provides clinical, demographic, and genetic characteristics of the asthmatic patient cohorts, stratified by their MEFV carrier status. There were no statistically significant differences between carrier and noncarrier patients with asthma with respect to age, sex, and onset of disease. However, carriers and noncarriers of MEFV mutations varied significantly in asthma severity scores, with carriers displaying less severe disease.

Table 3. Demographic and Clinical Variables Stratified According to MEFV Mutation Carrier Statusa Variable Age, mean ⫾ SD, y Male sex, No. (%) Age of disease onset, mean ⫾ SD, y Severity of asthma Mild intermittent Mild persistent Moderate persistent Severe persistent

Carriers (n ⴝ 11)

Noncarriers (n ⴝ 64)

P value

42.2 ⫾ 21.2 4 (36) 26.9 ⫾ 17.0

43.6 ⫾ 21.2 30 (47) 27.1 ⫾ 18

.80 ⬎.50 .98

1 7 3 0

6 8 35 15

a

⬍.002b

Data are given as number of subjects unless otherwise indicated. Mild groups vs moderate plus severe groups, as determined using the National Asthma Education and Prevention Program criteria for the severity of asthma.19 b

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DISCUSSION Genetic analysis of 75 patients with asthma and 45 unaffected first-degree relatives, coming from 46 families of Iraqi Jewish origin, revealed that the frequency of MEFV mutations in patients with familial asthma is lower than expected for the Iraqi Jewish population in Israel18 and is low compared with the rate of carriers in asymptomatic first-degree relatives of patients with asthma. Additional support, excluding a possible genetic diversion or a drift in the study population, comes from the distribution of the specific mutation type found in the families, which is altogether comparable to that described for a population of similar origin, dominated by V726A and E148Q mutations.18 Finally, the lower severity of asthma in carriers reinforces the concept of the present study (ie, MEFV mutations may protect from the development of asthma, at least in certain Mediterranean populations). The only finding that may cast doubt on these unequivocal results is the result of the subanalysis of 19 families that contributed both patients with asthma and unaffected relatives. In these families, a 2-fold difference in MEFV mutation carriage in favor of the unaffected relatives has not reached significance. However, when accounting for the number of mutated MEFV alleles, instead of the number of carriers, the differences between the groups remained significant, further supporting the protective role of MEFV in these families. The finding of 2 MEFV mutations in asymptomatic individuals (with regard to FMF manifestations), called FMF phenotype 3, is not an exception; on the contrary, it is the rule. Several population studies18,21,22 revealed that the frequency of phenotype 3 in the general population in Israel is higher than the frequency of overt FMF. The reasons for this paradox are multiple: low penetrance, mild symptoms, or low-grade inflammation as the sole manifestation of the disease. Our study suggests that the effects of these FMF alleles, either in a single or a double form, are far beyond the simplistic clinical presentation (FMF) that is perceived to be its main association. The high frequency of MEFV mutation carriers, found in the Iraqi Jewish population, in the present and earlier studies suggests that these DNA changes may confer biological advantage to people living in the Mediterranean basin. Several studies18,23 failed to find the culprit pathogen against which the protection is directed, implying it was eradicated some time in the past. However, as suggested by our findings in asthma, it is possible that the protective role of MEFV mutations is directed against the harmful effects (such as atopy and asthma) of the immune system in general, rather than against a certain specific microorganism. Our finding in asthma, however, is an exception to the general trend of MEFV mutation carriage, to prompt enhanced inflammation and functional impairment. For instance, in rheumatoid arthritis and multiple sclerosis, higher severity scores and faster deterioration to disability have been noted, respectively.24,25 Similarly, in patients with Crohn disease, extraintestinal manifestations and stricturing disease

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were significantly more common in MEFV mutation carriers.26 The only other disease toward which MEFV mutations seem to confer protection, to a limited degree, is systemic lupus erythematosus.27 An insight into these MEFV conflicting effects is basically still speculative. Several possible mechanisms may, however, explain the protection granted by carrying MEFV mutations. First, wildtype pyrin or marenostrin, the protein encoded by MEFV, is mainly expressed in neutrophils, monocytes, and eosinophils28 and is thought to play an inhibitory role in the intersection of apoptosis and inflammation, 2 allegedly opposite pathways, mediated by the apoptosis-associated specklike protein with a caspase recruitment domain.29 Based on the findings in the present study, we postulate that the mutated pyrin in both carriers and patients with FMF may divert the net effect of these pathways in bronchi toward increased apoptosis, counteracting the TH2-mediated reduced apoptosis of inflammatory cells in the bronchial tree characterizing asthma.30 –32 In accord with this hypothesis, a previous study33 showed enhanced apoptosis in patients with FMF. Second, the negative associations between asthma and MEFV mutations may originate from direct or indirect (eg, through interferon-␥) suppression by pyrin or marenostrin of TH2 activity, which propagates inflammation in asthma. Other atopic diseases, such as allergic rhinitis, also occur less than expected in patients with FMF, presumably because of a mechanism similar to that proposed in this study.34 Indeed, it is thought that TH1 polarization characterizes FMF.28,35 Our findings lend further support to this hypothesis. The significance of the study extends further to its sector (Iraqi Jewish) relevance. First, there are approximately 150,000 patients with FMF worldwide, and, therefore, assuming a 1 in 10 carrier rate in susceptible populations, there are more than 60 million MEFV mutation carriers living around the globe, to whom our finding might be pertinent. Second, given the central role of the pyrin domain in inflammation, our study highlights the need to look into this new asthma association. One way, perhaps, would be to conduct linkage analysis, using a candidate gene approach, focusing on proteins with a pyrin domain. In conclusion, the decreased frequency of MEFV mutations in patients with asthma of Iraqi Jewish origin and a mild phenotype in carriers of these mutations suggest that MEFV mutations may protect from asthma and perhaps underlie the reduced frequency of asthma, originally found by Danon et al,15 in the entire population with FMF. REFERENCES 1. Koppelman GH. Gene by environment interaction in asthma. Curr Allergy Asthma Rep. 2006;6:103–111. 2. Koppelman GH, Stine OC, Xu J, et al. Genome-wide search for atopy susceptibility gene in Dutch families with asthma. J Allergy Clin Immunol. 2002;109:498 –506. 3. Xu X, Fang Z, Wang B, et al. A genomewide search for quantitative-trait loci underlying asthma. Am J Hum Genet. 2001;69:1271–1277. 4. The Collaborative Study on the Genetics of Asthma (CSGA): a

520

5. 6.

7. 8. 9. 10. 11. 12.

13. 14. 15. 16. 17. 18.

19. 20. 21. 22. 23.

genome-wide search for asthma susceptibility loci in ethnically diverse populations. Nat Genet. 1997;15:389 –392. Ober C, Tsalenko A, Parry R, Cox NJ. A second-generation genome-wide screen for asthma-susceptibility alleles in a founder population. Am J Hum Genet. 2000;67:1154 –1162. Ober C, Cox NJ, Abney M, et al. Genome-wide search for asthma susceptibility loci in a founder population: the Collaborative Study on the Genetics of Asthma. Hum Mol Genet. 1998;7:1393–1398. Sohar E, Gafni J, Pras M, Heller H. Familial Mediterranean fever: a survey of 470 cases and review of the literature. Am J Med. 1967;43:227–253. The International FMF Consortium. Ancient missense mutations in new member of RoRet gene family are likely to cause familial Mediterranean fever. Cell. 1997;90:797– 807. The French FMF Consortium. A candidate gene for familial Mediterranean fever. Nat Genet. 1997;17:25–31. Gumucio DL, Diaz A, Schaner P, et al. Fire and ice: the role of pyrin domain-containing proteins in inflammation and apoptosis. Clin Exp Rheumatol. 2000;20(suppl 26):45–53. Bernot A, da Silva C, Petit JL, et al. Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever. Hum Mol Genet. 1998;7:1317–1325. Harton JA, Linhoff MW, Zhang J, Ting JP. Cutting edge: CATERPILLER: a large family of mammalian genes containing CARD, pyrin, nucleotide-binding, and leucine-rich repeat domains. J Immunol. 2002;169:4088 – 4093. Chamaillard M, Girardin SE, Viala J, Philpott DJ. Nods, Nalps and Naip: intracellular regulators of bacterial-induced inflammation. Cell Microbiol. 2003;5:581–592. Stehlik C, Reed JC. The PYRIN connection: novel players in innate immunity and inflammation. J Exp Med. 2004;200: 551–558. Danon Y, Laor A, Shlezinger M, Zemer D. Decreased incidence of asthma in patients with familial Mediterranean fever. Isr J Med Sci. 1990;26:459 – 460. Ozyilkan E, Simsek H, Telatar H. Absence of asthma in patients with familial Mediterranean fever. Isr J Med Sci. 1994;40: 237–238. Brenner-Ullman A, Melzer-Ofir H, Daniels M, Shohat M. Possible protection against asthma in heterozygotes for familial Mediterranean fever. Am J Med Genet. 1994;53:172–175. Kogan A, Shinar Y, Lidar M, et al. Common MEFV mutations among Jewish ethnic group in Israel: high frequency of carrier and phenotype 3 states and absence of a perceptible biological advantage for the carrier state. Am J Med Genet. 2001;102: 272–276. Expert Panel Report 2: National Institutes of Health Guidelines for the Diagnosis and Management of Asthma. Bethesda, MD: National Institutes of Health; 1997. NIH publication 97– 4051. Zaks N, Shinar Y, Padeh S, et al. Analysis of the three most common MEFV mutations in 412 patients with familial Mediterranean fever. Isr Med Assoc J. 2003;5:585–588. Stoffman N, Magal N, Shohat T, et al. Higher than expected carrier rates for familial Mediterranean fever in various Jewish ethnic groups. Eur J Hum Genet. 2000;8:307–310. Gershoni-Baruch R, Shinawi M, Leah K, Badarnah K, Brik R. Familial Mediterranean fever: prevalence, penetrance and genetic drift. Eur J Hum Genet. 2001;9:634 – 637. Ozen S, Balci B, Ozkara S, et al. Is there a heterozygote advantage for familial Mediterranean fever carriers? Clin Exp

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Rheumatol. 2002;20(suppl 26):S57–S58. 24. Rabinovich E, Livneh A, Langevitz P, et al. Severe disease in patients with rheumatoid arthritis carrying a mutation in the Mediterranean fever gene. Ann Rheum Dis. 2005;64: 1009 –1014. 25. Shinar Y, Livneh A, Villa Y, et al. Common mutations in the familial Mediterranean fever gene associate with rapid progression to disability in non-Ashkenazi Jewish multiple sclerosis patients. Genes Immun. 2003;4:197–203. 26. Fidder HH, Chowers Y, Lidar M, Sternberg M, Langevitz P, Livneh A. Crohn disease in patients with familial Mediterranean fever. Medicine. 2002;81:411– 416. 27. Ozen S, Bakkaloglu A. C reactive protein: protecting from lupus in familial Mediterranean fever [letter]. Ann Rheum Dis. 2005; 64:786 –787. 28. Centola M, Wood G, Frucht DM, et al. The gene for familial Mediterranean fever, MEFV, is expressed in early leucocyte development and is regulated in response to inflammatory mediators. Blood. 2000;95:3223–3231. 29. Martinon F, Hofmann K, Tschopp J. The pyrin domain: a possible member of death domain-fold family implicated in apoptosis and inflammation. Curr Biol. 2001;11:118 –120. 30. Mosmann TR, Sad S. The expanding universe of T-cell subsets:

Th1, Th2 and more. Immunol Today. 1996;17:138 –146. 31. Kunkel SL. Th1- and Th2-type cytokines regulate chemokine expression. Biol Signals. 1996;5:197–202. 32. Kay AB. Allergy and allergic disease. N Engl J Med. 2001;344: 109 –113. 33. Ozen S, Uckan D, Baskin E, et al. Increased neutrophil apoptosis during attacks of familial Mediterranean fever. Clin Exp Rheumatol. 2001;19:68 –71. 34. Sackesen C, Bakkaloglu A, Sekerel BE, et al. Decreased prevalence of atopy in paediatric patients with familial Mediterranean fever. Ann Rheum Dis. 2004;63:187–190. 35. Aypar E, Ozen S, Okur H, Kutluk T, Besbas N, Bakkaloglu A. Th1 polarization in familial Mediterranean fever. J Rheumatol. 2003;30:2011–2013.

Requests for reprints should be addressed to: Avi Livneh, MD Heller Institute of Medical Research Sheba Medical Center Tel Hashomer 52621, Israel E-mail: [email protected]

Answers to CME examination—Annals of Allergy, Asthma & Immunology, December 2007 Lanier B, Kai G, Marple B, Wall GM: Pathophysiology and progression of nasal septal perforation. Ann Allergy Asthma Immunol. 2007;99:473– 480. 1. c 2. d 3. d 4. c 5. c

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