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Dec 23, 2004 - 1Department of Gastroenterology, Rambam Medical Center, Haifa, Israel; ... Israel; 6Department of Pediatric, Gastroenterology and Nutrition, ...
Genes and Immunity (2005) 6, 134–139 & 2005 Nature Publishing Group All rights reserved 1466-4879/05 $30.00 www.nature.com/gene

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Prevalence and significance of mutations in the familial Mediterranean fever gene in patients with Crohn’s disease A Karban1, E Dagan2,3, R Eliakim1,4, A Herman2, S Nesher1, B Weiss5, D Berkowitz6, R Shamir4,6 and R Gershoni-Baruch2,4 1

Department of Gastroenterology, Rambam Medical Center, Haifa, Israel; 2Institute of Human Genetics, Rambam Medical Center, Haifa, Israel; 3Department of Nursing, Faculty of Welfare and Health Studies, University of Haifa, Israel; 4Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel; 5Division of Pediatric, Gastroenterology, Safra Hospital, Ramat-Gan, Israel; 6Department of Pediatric, Gastroenterology and Nutrition, Meyer Children’s Hospital, Rambam Medical Center, Haifa, Israel

The concurrence of Crohn’s disease (CD) and familial Mediterranean fever was repeatedly reported. In this study we determined the distribution and contribution of MEFV gene mutations to CD susceptibility and clinical heterogeneity. An Israeli cohort of 209 CD patients (120 men and 89 women) was investigated for mutations in the MEFV gene. A detailed chart review, interview and physical examination were used to determine sociodemographic and clinical characteristics. MEFV and NOD2/ CARD15 genotypes were analyzed in all patients and a genotype–phenotype correlation analysis was undertaken. The results of this study do not implicate MEFV mutations as major modifiers in CD. However, the E148Q MEFV variant was associated with susceptibility to perianal disease. More specifically, 19% (9/47) of CD patients with perianal disease carried the E148Q mutation compared to 6.7% (11/162) of CD patients without perianal involvement (OR 3.26, 95% CI 1.2–8.8, P ¼ 0.02). Although, for all mutations taken together, the prevalence of MEFV gene mutations among CD patients and controls was similar, the hypothesis that E148Q mutation modulates the phenotypic expression of CD is corroborated by the results of this study and needs to be further evaluated. Genes and Immunity (2005) 6, 134–139. doi:10.1038/sj.gene.6364156 Published online 23 December 2004 Keywords: Crohn’s disease; familial Mediterranean fever gene; NOD2/CARD15; genotype–phenotype correlation

Introduction Crohn’s disease (CD) is a chronic inflammatory bowel disease of unknown origin. Current data suggest that the interaction of genetic, immunologic and environmental factors lead to the chronic inflammation. A variety of different inflammatory mediators have a crucial role in the inflammatory process, attributed to an imbalance between proinflammatory and inhibitory cytokines. The recent discovery of NOD2/CARD15 as a susceptibility gene in CD1,2 and functional studies suggest that inappropriate responses to bacterial components may alter signaling pathways of the innate immune system, leading to the development and persistence of intestinal inflammation.3,4 NOD2/CARD15 mutations are present in 30–50% of Caucasian CD patients compared to 7–20% of healthy controls.3 Interestingly, having two copies of the risk alleles increases the risk for developing the disease 20- to 40-fold while having one copy confers a relatively small increased risk, indicating that NOD2/ CARD15 functions to a large extent as an autosomal Correspondence: Dr R Gershoni-Baruch, Department of Medical Genetics, Rambam Medical Center, Haifa 31096, Israel. E-mail: [email protected] Received 27 July 2004; revised 6 November 2004; accepted 7 November 2004; published online 23 December 2004

recessive trait. CD is a complex genetic disease and it is likely that multiple genes influence disease expression. Unfortunately, even if the association between the three main NOD2/CARD15 mutations (Arg702Trp, Gly908Arg and Leu1007fsinsC) and CD is clearly established, the additional genetic factors contributing to CD remain elusive. Other susceptibility loci suggest the involvement of additional genes, which alter homeostatic mechanisms and increase disease susceptibility.5 CD is common in Israel, with most studies reporting higher prevalence rates in Ashkenazi than non-Ashkenazi Jews.6 The association of CD with other, known genetically determined, disorders is well known. These associated diseases include Turner’s syndrome,7 and cystic fibrosis8 as well as complex autoimmune genetic diseases including ankylosing spondylitis (especially in conjunction with the HLA-B27 haplotype), psoriasis,9 and multiple sclerosis.10 Recently, Cattan et al11 reported a more frequent and more severe inflammatory bowel disease in French, non-Ashkenazi Jewish, families with at least one familial Mediterranean fever (FMF) patient. Fidder et al12 reported that CD appears to be more prevalent in individuals with FMF and presents later than in individuals without FMF. FMF is an inherited inflammatory disease common in populations of Mediterranean extraction.13 The disorder, noted for its clinical variability, is characterized by

MEFV gene and Crohn’s disease A Karban et al

recurrent episodes of fever, peritonitis, pleurisy, rashes and arthritis, and may be complicated by AA amyloidosis.13 FMF is caused by mutations in the gene MEFV, which encodes pyrin/marenostrin, a protein implicated in the regulation of neutrophil activity.14,15 Five sequence alterations in the MEFV gene identify the majority of FMF chromosomes.16–19 These include four conservative missense mutations (M680I, M694V, M694I, V726A), clustered in exon 10, which, together with mutation E148Q, in exon 2, account for the majority of FMF chromosomes found in our patients.20–22 The phenotypic variability of the disease is largely attributed to allelic heterogeneity. Mutation M694V and the complex V726AE148Q allele (V726A associated in cis with mutation E148Q) are associated with a severe phenotype and amyloidosis.23–28 Mutation M680I and V726A are associated with a moderate form of disease.28 Mutation E148Q, the most common variant found in our population, with an allele frequency of up to 10%, has reduced penetrance, and many individuals, homozygotes or compound heterozygotes for these mutations, remain symptom free.18,29,30 The E148Q variant was recently associated with AA amyloidosis and with obscure chronic inflammatory diseases suggesting that it may augment inflammation nonspecifically.31 Lately, some light was shed on the physiological role of pyrin/ marenostrin and its involvement in apoptosis and inflammation.32 Taken together with the observation that CD is seemingly more prevalent among patients with FMF,11,12,33 we set to determine the prevalence, distribution and contribution of mutations in the MEFV gene in patients with CD.

Table 1 Demographic and clinical characteristics of 209 patients

135

with CD Characteristics Demographic Gender n (%) Male Female

120 (57.4) 89 (42.6)

Ethnicity n (%) Ashkenazi North African Ashkenazi and North African Muslim Christian Arabs Druze

107 29 64 2 2 5

Clinical Age at diagnosis mean+s.d. (range) Smoking n (%) Family history n (%) (1st & 2nd degree) Type of CD n (%) 1. Inflammatory 2. Stricturing 3. Penetrating

(51) (13.9) (30.6) (1.0) (1.0) (2.3)

23.6+12.6 (1–68) 64 (30.6) 60 (28.7) 112 (53.6) 54 (25.8) 43 (20.6)

Extent n (%) 1. Terminal ileum 2. Colon 3. Ileocolon 4. Upper GI

70 28 94 17

Perianal involvement n (%) Extra manifestation n (%)Aa

47 (22.5) 53 (25.3)

(33.5) (13.4) (45) (8.1)

a

Results Demographic and clinical characteristics Table 1 summarizes the demographic and clinical data of 209 CD patients (120 males and 89 females; male/female ratio 1.35:1). The mean age at diagnosis of CD was 23.6712.6 years (range 1–64 years). In total, 107 patients (51%) were of Ashkenazi descent, 29 (13.9%) were North Africans Jews, 64 (30.6%) were of mixed Ashkenazi/nonAshkenazi Jewish descent and 9 (4.3%) were Israeli Arabs. One-third of the patients were smokers. Familial clustering of inflammatory bowel disease (first and/or second degree relative with IBD) was observed in 28.7% of patients. Of the 209 CD patients, 53.6% (112) manifested with inflammatory CD; 25.8% (54) with stricturing and 20.6% (43) with penetrating CD. Perianal disease occurred in 22.5% (47) and 25.3% (53) had extra gastrointestinal disease, mostly peripheral arthritis. MEFV mutant allele frequencies in CD patients and controls The distribution of the most frequent MEFV mutant alleles is depicted in Table 2. MEFV mutations do not emerge as major contributors to CD. The distribution of NOD2/CARD15 genotypes and MEFV mutations The frequencies of NOD2/CARD15 genotypes and MEFV mutant alleles are depicted in Table 3. Of the 209 CD patients, 71 (34%) carried one mutated NOD2/ CARD15 allele and 15 (7.2%) had two mutated alleles;

Includes arthritis/arthralgia, ankylosing spondylitis, skin disease, primary sclerosing cholangitis and eye diseases.

44 (21%) carried one mutated MEFV allele and 3 (1.4%) had two MEFV mutated alleles. The distribution of MEFV mutations was not significantly different in CD patients with different NOD2/CARD15 genotypes. Association between MEFV mutation carriage and CD phenotypic characteristics For all mutations taken together, no statistical clinical differences were found between carriers and noncarriers of MEFV gene mutations (Table 4). Excluding from the analysis, all CD cases who carried two mutated NOD2/ CARD15 alleles (n ¼ 15) yielded similar results. Association between NOD2/CARD15 mutation carriage and CD phenotypic characteristics Colon-only CD was significantly less prevalent among NOD2/CARD15 carriers (6/85; 7.1%) than noncarriers (22/121; 18.2%) (Po0.05). Other phenotypic characteristics (inflammatory type, small intestinal involvement, perianal disease, presence of extraintestinal manifestations) were not found to be associated to carriage of NOD2/CARD15 mutations. Association of E148Q mutation with clinical characteristics of CD patients A significant association was found between E148Q mutation carriage and perianal disease (Table 5). More Genes and Immunity

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Table 2 Contribution of MEFV gene mutations to Crohn’s disease Ethnicity

Mutationa

Ashkenazi Jews CD patients Controls

V726A

Moroccan Jews CD patients Controls

M694V

Mixed (Ashkenazi & non-Ashkenazi) CD patients Controls

V726A/M694V

All CD cohort CD patientsb Controlsc

E148Q

n

Carriers, n (%)

OR

95% CI

511 107 404

10 (9.3) 30 (7.4)

1.285 1.0

0.607–2.720

272 29 243

5 (17) 27 (11)

1.67 1.0

0.587–4.730

446 64 382

8 (12.5) 34 (8.9)

1.462 1.0

0.644–3.321

689 209 480

21 (10) 65 (13.5)

0.713 1.0

0.423–1.201

a

Data analysis is depicted for the most prevalent MEFV mutations. Includes nine CD patients of Arab descent. c In total, 480 control samples were differentially tested according to ethnicity. b

Table 3 Distribution of NOD2/CARD15 genotypes and the different MEFV mutations in 209 CD patients NOD2/CARD15 genotypea

MEFV mutations Carriers/no. tested (%)b E148Q

M694V

V726A

M694I

M680I

K695R

Wild type (/) Heterozygote (+/) Homozygote/compound heterozygote (+/+)

11/123 (8.9) 8/71 (11.3) 2/15 (13.3)

4/62 (6.4) 3/33 (9.1) 0/7 (0)

10/102 (10) 2/56 (3.6) 4/13 (30.7)

0/6 (0) 0/3 (0) 0/0 (0)

1/2 (50) 0/0 (0) 0/0 (0)

3/100 (3) 2/56 (3.4) 0/12 (0)

Total P-value

21/209 (10) NS

7/102 (6.9) NS

16/171 (9.3) NS

0/9 (0) NS

1/2 (0) NS

5/168 (3) NS

a

Wild type (/) for any of the three common NOD2/CARD15 mutations, heterozygote (+/) for any of the three mutated alleles; homozygote and/or compound heterozygote (+/+). b MEFV mutations were differentially investigated according to ethnicity. Three patients were MEFV compound heterozygotes (E148Q/M694V, E148Q/K695R and V726A/K695R). NS ¼ not significant.

Table 4 Clinical characteristics of 209 CD patients related to MEFV carrier state Clinical characteristics

Age at diagnosis (mean+s.d.) Gender (male) n (%) Inflammatory type n (%) Small intestinal involvement n (%) Perianal disease n (%) Extraintestinal manifestation n (%) Family history n (%) (1st & 2nd degree)

MEFV carriers 47 (22.5%)

MEFV noncarriers 162 (77.5%)

24.3+12.4 27 (57.4) 27 (57.4) 40 (85) 13 (27.6) 12 (25.5) 12 (25.5)

23.4+12.7 93 (57.4) 85 (52.5) 141 (87) 34 (21) 41 (25.3) 48 (29.6)

specifically, 21.2% (10/47) of CD patients with perianal disease carried the E148Q mutation compared to 6.7% (11/162) of those who did not have perianal disease (Po0.01). This observation remains significant using the logistic regression model (OR 3.26; 95% CI 1.2–8.8) and after excluding from the analysis patients who carry two mutated NOD2/CARD15 alleles (n ¼ 15). Genes and Immunity

Statistical tests

t ¼ 0.340; P ¼ 0.65 w ¼ 0; P ¼ 0.5 w2 ¼ 0.3; P ¼ 0.577 w2 ¼ 0.133; P ¼ 0.439 w2 ¼ 1.2; P ¼ 0.549 w2 ¼ 0.21; P ¼ 0.9 w2 ¼ 0.263; P ¼ 0.608

Discussion To examine the recently described association between CD and FMF and to address the proposed notion that disease severity and age of onset in CD may be modulated by the presence of MEFV mutations,11,12 we determined the distribution of MEFV gene mutations in

MEFV gene and Crohn’s disease A Karban et al

Table 5 Multivariate analysis in 209 CD patients: association between E148Q carriage and the phenotypic variables Clinical phenotypes Perianal disease Extraintestinal manifestation Inflammatory type Small bowel involvement Family history

OR

95% CI

P

3.257 0.939 1.344 0.746 0.610

1.200–8.837 0.331–2.666 0.501–3.605 0.221–2.513 0.191–1.945

0.020 0.906 0.558 0.636 0.403

an Israeli CD cohort. MEFV, which encodes pyrin/ marenostrin, was recently found to play a pivotal role in the inflammation/apoptosis cascade,32 a cascade seemingly relevant to the pathogenesis of CD. The MEFV gene is located at the short arm of chromosome 16 and not within the IBD1 locus. However, stratification of IBD1 based on NOD2/CARD15 genotype provided evidence for a second IBD gene located on chromosome 16p.34,35 NOD2/CARD15 belongs to the recently described family of intracellular Nod proteins, which contain a nucleotide-binding site domain flanked by a leucine-rich repeat (LRR) domain that is important for bacterial binding. NOD2/CARD15 shares sequence homology in the nucleotide-binding and LRR domains with a wider family of B20 proteins, involved in apoptosis and inflammation, collectively known as the CATERPILLER (‘CARD transcription enhancer, R [purine]-binding, pyrin, lots of leucine repeats’) family. The MEFV encodes pyrin/marenostrin.36 Pyrin/marenostrin contain an Nterminal domain that encodes a death domain-related structure, now known as the pyrin domain, or PyD. Synthesis of the available data indicates that both pyrin and cryopyrin (involved in periodic fevers other than FMF) interact via their PyDs with a common adaptor protein, ASC. ASC itself participates in at least three important cellular processes: apoptosis, recruitment and activation of pro-caspase-1 (with associated processing and secretion of IL-1beta), and activation of NF-kappaB (a transcription factor involved in both initiation and resolution of the inflammatory response). Through PyD:PyD interactions, pyrin appears to modulate the activity of all three of these processes, each of which plays a crucial role in the inflammatory pathways that characterize the innate immune system.32 At least some of these inflammatory pathways are relevant to the pathogenesis of CD. This study compared the frequency of MEFV mutant alleles in a CD affected vs an ethnically matched control population. Carriers of MEFV mutations are equally prevalent in CD patients and controls. MEFV alleles do not seem to play a major role in disease susceptibility for CD and no evidence of epistasis between NOD2/ CARD15 and MEFV genes was found. Three CD patients who carried two MEFV mutated alleles did not display clinical signs compatible with FMF. This is attributed to the fact that they carried extremely mild, rather nonpenetrant, MEFV mutations (M694V/E148Q; V726A/K695R; E148Q/K695R). The notion that not only susceptibility to CD but also disease location and behavior may be influenced by

genetic determinants has stimulated the search for disease modifying genes. To date, in addition to the NOD2/CARD15 gene, most efforts have focused on HLA genes and genes involved in the regulation of the immune response and maintenance of mucosal integrity. For example, the DRB1*03 allele seems to protect from perianal disease and HLA DR7 was found to be associated with ileal rather than colonic disease.37 In our study, E148Q was found to be more common in CD patients with perianal disease. The significance of the E148Q mutation in FMF is controversial. Its prevalence is similar in FMF patients and control subjects. Furthermore, it contributes to the pathogenesis of FMF predominantly when coupled with other, presumably more disruptive, MEFV mutations.38 Since 10% of our population do carry the E148Q variant,21 and in view of the fact that the Israeli population is not considered to be overwhelmingly disease prone, one should consider the E148Q variant as contributory to chronic inflammation only in genetically susceptible individuals. Booth et al31 reported that healthy carriers of the FMF trait had modest but significantly elevated baseline plasma levels of the classical acute-phase reactant, Creactive protein, suggesting that their general response to inflammatory stimuli may be upregulated.39,40 The acutephase response to tissue damage is highly conserved among species, and in evolutionary terms is likely to be beneficial, probably by augmenting innate host resistance to infection. On the other hand, an intense prolonged acute-phase response, in carriers of E148Q mutation, may interact with a host of additional genetic and environmental factors and may predispose to perianal disease. Extensive heterogeneity is observed in the current cases classified as CD, which likely represent a heterogenous subset of disorders, with differing pathogenic mechanism. In multiple studies, a significant association was found between ileal and stricturing disease and the carriage of one or more NOD2/CARD15 risk variant alleles.41–44 In particular, double-dose carriers are relatively uncommonly observed in colon-only CD. This study confirms that colon-only CD is relatively rare among NOD2/CARD15 carriers. Perianal disease was not shown to be associated with carrying NOD2/ CARD15 risk alleles. The association of the E148Q variant with perianal disease is intriguing in this aspect. Our results suggest that this variant may play a role in the pathogenesis of CD phenotype that is not associated with NOD2/CARD15. This hypothesis needs further study with much larger sample size.

137

Methods Patients and controls Patients. A cohort of 209 patients, with CD, was recruited between 2001 and 2003 from two medical centers in Israel: Rambam Medical Center in Haifa and Safra Hospital in Ramat-Gan. All subjects signed an informed consent form allowing the use of their DNA for genetic research purposes. The study was approved by the two centers Institutional Review Board. Sociodemographic and clinical profile (age at diagnosis, ethnicity, smoking behavior, family history, etc) were determined from medical records, questionnaires Genes and Immunity

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and interviews. Ashkenazi and Sepharadic Jewish ethnicities were carefully assigned on the basis of the birthplace of the four grandparents. Patients were considered smokers if they smoked at least seven cigarettes per week, at least 1 year, anytime during their life. An appropriately trained gastroenterologist confirmed all diagnoses of CD by first hand review of endoscopic, pathology and radiology reports and operative notes. More specifically, the diagnosis of CD used established criteria.45,46 The disease extent and behavior were determined using the Vienna Classification.47 Briefly, patients were considered to have penetrating disease if they documented occurrence of intra-abdominal or perianal fistulas, inflammatory masses and/or abscesses at any time in the course of the disease excluding postoperative intra-abdominal complications. Stricturing disease was defined as the occurrence of constant luminal narrowing with prestenotic dilatation or obstructive signs/symptoms without the presence of penetrating disease. By exclusion, all other CD patients were defined as having an inflammatory disease. Disease location was classified as terminal ileal, colonic, ileocolonic and upper-gastrointestinal (disease limited proximal to the ileum). At least, both a small and a large bowel examination were required for classification. Controls. The prevalence of MEFV mutations was previously established in a group of 480 healthy individuals, from different ethnic descent, who presented for heterozygote detection of recessive diseases.21 Genetic analysis DNA extraction. DNA was extracted from whole blood using the Quiagen Genomic DNA purification kit according to the manufacturer’s protocol. MEFV mutation analysis. Six predominant mutations in the MEFV gene (E148Q, M694V, V726A, M694I, M680I and K695R) were investigated by PCR amplification followed by digestion with appropriate enzymes made to distinguish the wild-type allele from the mutant allele, as previously described.18,48 Two different M680I mutations (either a or b) abolish a constitutive Hinf-I restriction site and cannot be distinguished by our assay. Based on the differential distribution of MEFV mutations in the diverse ethnic groups,21 Ashkenazi Jews were selectively studied for mutations V726A, K695R and E148Q; North-African Jews for M694V and E148Q; Mixed Ashkenazi/non-Ashkenazi for M694V, V726A, K695R and E148Q and Israeli Arabs for M694V, M694I, M680I, V726A and E148Q. NOD2/CARD15 mutations analysis. The Arg702Trp and Leu1007fsinsC mutations were investigated using single tube allele-specific PCR and for Gly908Arg using restriction enzyme digestion assay as previously described.1,49 Data analysis The prevalence of MEFV mutations in CD patients and ethnically matched controls was compared and the contribution of MEFV gene mutations to CD was examined using OR and 95% confidence interval.

Genes and Immunity

The CD cohort was then subdivided according to NOD2/CARD15 genotype (wild type for one of the three mutations, /; heterozygote for one of the three mutations / þ and homozygote or compound heterozygote, þ / þ ). Each group was compared as regards the prevalence of the different MEFV mutations. The significance of differences between groups was calculated by either the w2 statistics or Fisher’s exact test for categorical data or the t-test for quantitative data. The phenotypic characteristics of CD were further analyzed in relation to carriage of MEFV and NOD2/ CARD15 mutations taken together and separately. Patients with penetrating and stricturing CD were compared to patients with inflammatory CD and patients with small bowel involvement were compared to patients with colon-only disease. A logistic regression model was used to test for a significant association between different MEFV genotypes and phenotypic variables of CD (small bowel involvement, perianal disease, inflammatory behavior, extraintestinal manifestation and family history for CD). To control for potential confounding effects due to ethnic variation, ethnicity was entered as a covariate variable. This test was also used to show whether the association between MEFV variants and phenotype (eg perianal disease) was primary or secondary to other related phenotypes (eg small bowel involvement).

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