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Evaluation of common mutations in the Mediterranean fever gene in Multiple Sclerosis patients: Is it a susceptibility gene?
Journal of the Neurological Sciences 294 (2010) 38–42
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Evaluation of common mutations in the Mediterranean fever gene in Multiple Sclerosis patients: Is it a susceptibility gene? Aysun Unal a,⁎, Ahmet Dursun b, Ufuk Emre a, Nida F. Tascilar a, Handan Ankarali c a b c
Zonguldak Karaelmas University, Faculty of Medicine, Department of Neurology 67700, Kozlu, Zonguldak, Turkey Zonguldak Karaelmas University, Faculty of Medicine, Department of Medical Genetics 67700, Kozlu, Zonguldak, Turkey Zonguldak Karaelmas University, Faculty of Medicine, Department of Bioistatistics 67700, Kozlu, Zonguldak, Turkey
a r t i c l e
i n f o
Article history: Received 17 December 2008 Received in revised form 13 March 2010 Accepted 20 April 2010 Available online 18 May 2010 Keywords: Familial Mediterranean fever MEFV gene Pyrin domain Multiple Sclerosis Susceptibility Inflammation
a b s t r a c t Purpose: Multiple Sclerosis (MS) is a disease of the central nervous system characterized by multiple areas of inflammation and demyelination in the white matter of the brain and spinal cord. MEFV gene, which is the main factor in familial Mediterranean fever, is an intracellular regulator of inflammation. This study was designed to determine if known mutations in pyrin domain of MEFV gene are involved in MS and associated with MS morbidity. Methods: Fifty-three patients with MS and 66 healthy subjects, who were all Turkish, were included in this study. Five pyrin gene mutations (E148Q, M680I, M694V, M694I and V726A) were detected in the patients and controls by using the PRONTO™ FMF Basic Kit according to the manufacturer's instructions. Results: Pyrin gene mutations were found in 20 of the 53 MS patients (38%) and in seven of the 66 healthy subjects (11%). The frequency of total pyrin domain mutations was significantly higher in the MS patients than in the healthy subjects (p b 0.0001). The frequencies of M694V, E148Q and V726A mutations were significantly higher in the patients than in the healthy subjects (p = 0.02, p = 0.013, p = 0.004 respectively). The mean time to reach EDSS score 3.0 was earlier in the patients with MEFV gene mutation (p = 0.02) and the relapse rate was slightly higher among the MS patients carrying MEFV gene mutation (p = 0.04). Conclusion: The results of this study supported the hypothesis that MS patients with MEFV mutation seem to have the susceptibility to develop a more progressive disease. Moreover, these data suggest that MEFV mutations may increase the risk of MS development. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. The FMF gene (MEFV) was identified in 1997 by positional cloning [1]. According to INFEVERS (http://fmf.igh.cnrs.fr/infevers/) many MEFV mutations have been identified so far [2]. Five founder mutations V726A, M694V, M694I, M680I and E148Q account for 74% of FMF chromosomes from typical cases (Armenians, Arabs, Jews, and Turks) [3]. MEFV gene locus has been mapped to chromosome 16p 13.3 [1,4]. This gene encodes a 781-amino-acid protein named as pyrin. Pyrin is predominantly expressed in polymorphonuclear leukocytes (PMNs) and cytokine-activated monocytes [4,5]. Pyrin consists of four functional domains, a B-box zinc-finger domain, a coiled coil domain, a C-terminal B30.2 domain, and a 92-amino-acid N-terminal pyrin domain that is shared by a number of other proteins involved in apoptosis and inflammation [6]. The pyrin domain is a member of the
⁎ Corresponding author. Tel.: +90 5364428655; fax: +90 3722610155. E-mail address: [email protected] (A. Unal). 0022-510X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2010.04.008
six-helix bundle, death-domain superfamily that includes death domains (DD), death effector domains (DED), and caspase recruitment domains (CARDs) [7,8]. Thus, in patients with lack of pyrin protein (or its activity) due to hereditary defects, there is no suppression or inhibition of the inflammatory process, thereby leading to a full-blown attack [9]. Although FMF is a multisystem disease, its relationship with CNS involvement is not certain. Neurological complications during the course of FMF such as aseptic meningitis, pseudotumor cerebri, and nonspecific EEG abnormalities during febrile attacks have been previously reported [10,11]. Polyarteritis nodosa, microscopic polyangiitis and Henoch–Schonlein purpura are the three vasculitic diseases that have been known to be related with FMF. Diseases that have been proposed to be associated with FMF include Behcet's Disease (BD), inflammatory bowel disease and juvenile chronic arthritis [12–14]. These related diseases may also cause neurological complications. CNS demyelinating lesions in FMF patients have been reported rarely. Multiple Sclerosis (MS) is a pathogenically complex disease associated with a profound heterogeneity in the structural and immunopathologic features of the lesions, which vary depending on
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the stage of demyelinating activity or phase of the disease (early or late). In vitro and in vivo data indicate that multiple immune effector mechanisms can potentially lead to inflammation, focal demyelination, and tissue injury in MS [15,16]. A prospective search of MS in a small cohort of FMF cases remained negative [17]. In a previous study of a Jewish cohort with MS increasing disability occurred more rapidly in patients carrying the M694V allele of MEFV [18]. The pathophysiological relation between FMF and MS is still in debate. When combined with a Th1 related autoimmune disease, carrying a MEFV gene mutation may aggravate the disease course, as shown for carriers among patients with rheumatoid arthritis [19]. Also of note is the finding that healthy heterozygotes for MEFV mutations appear to have higher than normal blood levels of acute phase reactants, supporting the role of MEFV mutation in the induction of inflammation [20]. In this study, we aimed to determine whether mutations of pyrin domain of MEFV gene in Turkish MS patients are more prevalent than healthy controls and if they have an impact on the clinical disease course of MS. 2. Materials and methods Fifty-three MS patients and 66 healthy subjects were included in this study. The MS patients were followed-up on a regular basis in every 3–6 months at the Neurology outpatient-clinic of Zonguldak Karaelmas University, Faculty of Medicine. Informed consent was obtained from all the patients that participated in this study, and the local ethics committee of Zonguldak Karaelmas University, Faculty of Medicine approved the study. The Kurtzke Expanded Disease Status Scale (EDSS) scores of the patients in the follow-up examinations and all the clinical and demographic data of the patients were retrospectively obtained from the hospital computer database. The Progression index (EDSS/ duration of disease [years]) was determined in each patient included in the study. Blood samples of the MS patients were collected at the Medical Genetics Department of Zonguldak Karaelmas University, Faculty of Medicine. Genomic DNA was extracted from the peripheral blood leucocytes using the E.Z.N.A® Blood DNA Isolation Kit (Cat. No: D3392-02) according to the manufacturer's instructions. Five pyrin gene mutations (E148Q, M680I, M694V, M694I and V726A) were determined in the patients and controls by using the PRONTO™ FMF
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Basic Kit (Sayvon Diagnostic Ltd. Cat. No. 9904) according to the manufacturer's instructions. Descriptive statistics were computed as Mean ± SD and frequencies (count and percent). Prevalence of different MEFV gene mutations in the MS patients and healthy subjects was compared by the Fisher's exact chi-Square test. Pearson chi-square test was used to analyze the relation between categorical clinical and/or prognostic features of mutation carriers and non-carriers. Student t-test was preferred to analyze differences between the two groups about continuous clinical and/or prognostic features. Kaplan–Meier curve was drawn to present the probability of MS patients to reach EDSS: 3.0 or 6.0 during the disease course in relation to the MEFV genotype. p b 0.05 was considered statistically significant. SPSS (ver. 11.0) program was used for all the statistical analysis.
3. Results Between 2002 and 2008, a total of 90 cases with demyelinating disorders were registered in our clinic. Among these, 64 had definite MS; 16 had possible MS (mostly clinically isolated syndromes), and the remaining had other demyelinating diseases (acute disseminated encephalomyelitis, Devic's disease, recurrent optic neuritis, tumorlike or other unclassified demyelinating disorders). Fifty-three definite MS patients fulfilling Mc Donald's criteria were included in this study [21]. Eleven definite MS patients were excluded due to inadequate follow-up data. Thirty-six of the included 53 patients were female, and the mean age of the patients was 37 years. The mean follow-up time was 2.8 ± 1.7 years. None of these patients had clinical findings compatible with a systemic vasculitis. Screening of all the patients for systemic findings suggestive of BD was negative. Serologic screening tests for collagen vascular diseases (anti nuclear antibodies, antibodies against extractable nuclear antigens, antineutrophilic cytoplasmic antibodies, antiphospholipid antibodies and tests for syphilis) were all negative. One MS patient fulfilled modified Tel Hashomer criteria for diagnosis of FMF [22]. Fifty-two of the MS patients were not symptomatic for FMF and except one patient, none of the patients had a family history of FMF. The controls (n = 66) were all healthy subjects and had no symptoms for either FMF or MS. Fourteen of the controls were female, and the mean age of the controls was 34 years.
Table 1 Neurological and laboratory characteristics of MS patients carrying MEFV gene mutations (F: female, M: male, ON: optic neuritis, TM: transverse-myelitis, BS: brainstem, C: cerebellar, RR: relapsing–remitting, SP: secondary progressive, PP: primary progressive, PI: progression index, N: normal, ND: not determined, OB: oligoclonal band). Case no/sex
MEFV gene mutations
Age at onset (years)
Onset symptom
Disease duration (years)
Course
Outcome (final visit)
OB
1/F 2/F 3/F (Fig. 1a) 4/F (Fig. 1g–h) 5/M 6/M 7/F (Fig. 1f) 8/F 9/F (Fig. 1c) 10/F (Fig. 1e) 11/F 12/F 13/F 14/F 15/M 16/M 17/F (Fig. 1d) 18/F 19/M 20/F (Fig. 1b)
M694V /M694V V726A/V726A M694V/– V726A/– E148Q/– E148Q/– M694V/V726A E148Q/– E148Q/– M694V/– M694V/– M694I/– E148Q/– M694V/– M694V/– E148Q/– M694V/– V726A/– M680I 694V/E148Q
40 28 30 39 32 49 20 30 34 27 23 29 35 33 38 37 22 33 47 45
ON TM BS ON BS Pyramidal BS ON Pyramidal BS ON ON Sensorial ON Pyramidal BS BS ON BS Pyramidal
6 10 3 5 2 8 3 9 8 4 12 20 8 2 10 1 4 7 2 7
SP SP SP RR RR SP RR RP RR RR RR SP RR RR RP RR RR PP RR SP
EDSS:6.5 PI: 1.08 EDSS: 7.0 PI:0.70 EDSS:6.0 PI:2.00 EDSS:3.0 PI:0.38 EDSS:2.0 PI:0.38 EDSS:3.0 PI:1.07 EDSS:1.0 PI:0.57 EDSS:3.5 PI:0.60 EDSS:4.0 PI:1.0 EDSS:1.0 PI:0.33 EDSS:1.0 PI:0.50 EDSS:7.5 PI:0.25 EDSS:1.5 PI: 0.08 EDSS:1.0 PI:0.19 EDSS:2.5 PI:0.50 EDSS:1.0 PI:1.0 EDSS:1.0 PI:0.25 EDSS:4.0 PI:0.50 EDSS:1.0 PI:0.39 EDSS:7.5 PI:0.25
(+) (+) (+) (+) (−) ND (+) (+) (+) (+) ND (+) ND (+) (−) (+) (−) ND (−) (+)
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A. Unal et al. / Journal of the Neurological Sciences 294 (2010) 38–42
Table 2 Frequency of common MEFV mutations among MS patients and healthy subjects. Mutation type
MS patients n/2n
Healthy subjects n/2n
p value
M694V E148Q M680I V726A Total
11/106 7/106 1/106 5/106 24/106
4/132 1/132 2/132 0/132 7/132
0.02 0.013 0.691 0.004 b0.0001
Pyrin gene mutations were found in 20 of the 53 MS patients (38%) (fifteen female and five male) (Table 1). One patient was homozygous for M694V and one patient was homozygous for V726A, whereas two patients were carrying two different mutations (M694V/V726A; M694V/E148Q). M694V mutation was recorded in ten; E148Q in seven, V726A in four, and 680I in one case. There was no patient with M694I mutation. In the control group, MEFV mutations were found in seven of the 66 healthy subjects (11%). Four of 66 (6%) controls had M694V mutation, one (2%) had E148Q mutation and two (3%) had M680I mutation. The frequency of total pyrin gene mutations was significantly higher in the MS patients than in the healthy subjects (p b 0.0001). The frequency of M694V, E148Q and V726A mutations was found significantly higher from healthy subjects (p = 0.02, p = 0.013, p = 0.004 respectively). The frequency of the M680I mutation was not significant (Table 2). In order to determine whether the presence of MEFV gene mutation could aggravate the clinical expression of MS, we compared the demographic and clinical variables of mutation carriers and noncarriers. No significant differences were found between the two groups for mean age, average disease duration, and the mean age at onset. The course of disease was Relapsing–Remitting (RR) in 11 (55%), Relapsing Progressive (RP) in 2 (10%), Secondary Progressive (SP) in 6 (30%) and primary progressive (PP) in one patient (5%) in carriers. RR course was seen in 23 (69.7%); RP course, in 2 (6.1%); SP course, in 7 (21.2%) and PP course in one patient in non-carrier group. No statistically significant differences were found between the two groups for disease course (p = 0.754) and symptoms of MS onset (p = 0.381). Mean EDSS score (in the last neurological examination) was 3.2 ± 2.4 in carriers and 2.1 ± 2.1 in non-carriers (median scores 2.75 and 1.5 respectively). There were five patients (25%) in carriers and three
Table 3 Clinical and prognostic features of MS patients carrying a common MEFV gene mutation (ON: optic neuritis, TM: transverse-myelitis, BS: brainstem symptoms, RR: relapsing– remitting, SP: secondary progressive, PP: primary progressive, ⁎:significant p-value).
Age Disease duration (years) Age at onset Disease course
Disease onset symptom
RR RP SP PP ON TM BS Pyramidal Sensorial Polysymp. Cognitive
Patients reaching EDSS: 3 Patients reaching EDSS: 6 Mean time to reach EDSS 3 (years) Mean time to reach EDSS 6 (years) Relapse rate in RRMS patients
Carriers
Non-carriers
p value
40 ± 8.5 6.6 ± 4.5 33.6 ± 7.9 11 (55%) 2 (10%) 6 (30%) 1 (5%) 7 (35%) 1 (5%) 7 (35%) 4 (20%) 1 (5%) – – n = 10 n=5 3.9 ± 1.6 6.2 ± 5.0 1.6 ± 1.1
36 ± 9.2 7.1 ± 5.9 29.1 ± 9.4 23 (69.7%) 2 (6.1%) 7 (21.1%) 1 (3%) 7 (21.1%) 7 (21.1%) 6 (18.2%) 7 (21.1%) 4 (12.1%) 1 (3%) 1 (3%) n = 12 n=3 7.6 ± 4.3 11.7 ± 9.1 1.1 ± 0.9
p = 0.133 p = 0.711 p = 0.062 p = 0.754
p = 0.381
p = 0.329 p = 0.137 p = 0.02* p = 0.303 p = 0.04*
patients (9.1%) in non-carriers whose EDSS scores were ≥6.0. The mean progression index was 0.6 ± 0.4 in the carriers and 0.4 ± 0.4 in non-carriers. In the carrier group, the rate of the patients with progression index ≥ 0.5 was 55% (n = 11) and in the non-carrier group, 24% (n = 8). Although the mean progression index scores of the carriers seem to be higher, the difference was not statistically significant (p = 0.103). The comparison of the mean times to reach EDSS score 3.0 showed that it was earlier in the patients with MEFV gene mutation (p = 0.02). The mean time to reach EDSS = 3.0 was 4 years in the carriers and 7.5 years in the non-carriers. The mean time to reach EDSS = 6.0 was 5 years vs 13.5 years, respectively. No significant differences were determined between the two groups for the time required to reach EDSS score 6.0 (Table 3). The statistical analysis performed after the exclusion of primary progressive cases revealed no differences between the carriers and non-carriers for age, disease duration, progression index, frequency of the patients with EDSS ≥ 3.0, and the mean time to reach EDSS = 3.0. There were no patients with EDSS ≥ 6.0. The only statistically significant result was that the annual relapse rate was slightly higher among MS patients carrying MEFV gene mutation (p = 0.044). MEFV 694V was the most frequent mutation type in our study; therefore, we compared the mean times to reach EDSS: 3.0 and 6.0 of M694V mutation carriers with the other mutation type carriers (E148Q or M680I or V726A) and non-carriers. We found no differences between the progression milestones of the three groups. In all patients Magnetic Resonance Imaging (MRI) scans revealed typical MS lesions fulfilling the MRI criteria suggested by Mc Donald et al. [21]. Demyelinating lesions with nodular or ring-like enhancement (Fig. 1) were present in ten of the 20 MEFV gene mutation carrier patients. Spinal cord lesions were detected in fourteen of the carriers. Sixteen of the patients with MEFV gene mutation were subjected to CSF examination. The CSF samples were acellular with normal protein and glucose levels. Oligoclonal IgG bands were detected in twelve of the sixteen patients (Table 1). 4. Discussion In the present study, MS patients showed an increased frequency of MEFV (M694V, E148Q V726A and M680I) mutations compared to unaffected ethnically matched controls (38% and 11%). The frequency of M694V, E148Q and V726 mutations individually was also found to be significantly higher (Table 2). Shinar et al. studied mutations in the MEFV gene in MS patients in 2003 in Israel. They reported that the combined frequency of three founder mutations M694V, E148Q and V726A, and each mutation alone showed no increased frequency in MS patients. In their statistical analysis, the mutations in their study seemed to have affected the disability and relapse rate of the disease in the non-Ashkenazi MS patients group [18]. When we used Kaplan– Meier curve analysis, we also found that mutation carriers showed a statistically significant decrease in the mean time to reach EDSS: 3.0 compared to non-carriers. The mean time to reach EDSS: 6.0 also seemed to be earlier in the carriers, but it was not statistically significant. In accordance with the results of the study by Shinar et al., the annual relapse rate was slightly higher among RRMS patients carrying MEFV gene mutation. To date, numerous studies evaluated the degree of correlation between genotype and phenotype among patients with FMF. These studies, comparing disease phenotype with MEFV genotype, have indicated that some mutations are more pathogenic than others, but that clinical expression of the disease can vary widely among patients with the same mutations [23,24]. In the populations most typically affected by FMF, the mutation that appears to disrupt the function of pyrin to the greatest extend encodes the M694V variant [25,26]. M694V mutation is not only associated with a more severe clinical picture of FMF, but also confers a higher risk for these patients developing AA amyloidosis. In our study group, two MS patients were
A. Unal et al. / Journal of the Neurological Sciences 294 (2010) 38–42
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Fig. 1. a) Axial flair MR imaging of patient 3 with periventricular ovoid hyperintense lesions b) T1 axial MR image of patient 20 with hypointense periventricular lesions (black-holes) and brain atrophy c) Sagittal T1 MR imaging of patient 9 with ring and nodular gadalinum enhancing periventricular lesions d) Axial T1 MR imaging of patient 17 with open-ring, ring and nodular gadalinum enhancing lesions e) Coronal T2 brainstem MR imaging of patient 10 with hyperintense lesions in pons, medulla-oblongata and spinal cord f) Axial T2 MR imaging of patient 7 with ponto-cerebellar hyperintense lesions g) Sagittal T2 cervical spinal MR imaging of patient 4 with C6 localized hyperintense lesion h) same lesion on axial T1 MR imaging with gadalinium enhancement.
homozygous for MEFV gene mutation. Of these patients, one patient was homozygous for M694V, and one was homozygous for V726A. The first one with diagnosis of secondary progressive MS, fulfilled modified Tel Hashomer criteria for diagnosis of FMF, and she responded well to immunosuppressive therapy with colchicum. She was operated three times with the diagnosis of appendicitis, inguinal hernia, and over torsion because of recurrent abdominal pain in her twenties. The family history of the patient revealed abdominal pain attacks in her father; who died at the age of 43 with no diagnosis. The patient also described skin lesions and arthritis in her wrists and knees resolving in a week for several years [27]. Furthermore, some individuals may have paired pathogenic MEFV mutations but do not have clinical features of FMF [3]. The case of the second MS patient with homozygous MEFV V726A mutation supports studies suggesting that V726A homozygotes have milder forms [28]. She had no clinical finding that may be associated with FMF. She presented with a secondary progressive MS with no response to immunomodulatory therapy and mitoxantrone. Shinar et al., in their study on MS patients, have reported that progression to disability was earlier in carriers of the M694V mutation [18]. In this study, we also found that MEFV 694V mutation was the most frequent type; however, there were no differences between the progression milestones of the different mutation carriers. The diagnosis of FMF still remains predominantly clinical since mutations are not always penetrant and cannot always be identified on both alleles [29]. Paired MEFV mutations were detected in 84% of FMF patients and single mutations in 12%. Mutations in the MEFV gene may also be involved in non FMF disorders [28,30,31]. Recent studies from Turkey reported the frequency of MEFV mutation carriers as 10%–25% in Turkish population [14,24,32]. We found the frequency of MEFV mutations in 11% of healthy individuals, and most of the mutation carrier MS patients were heterozygous. The genetic defect in FMF was proposed to be the impaired control of inflammation in response to certain recognized or unrecognized
stimuli. Indeed chronic inflammatory disorders such as vasculitidis and Behcet's disease were reported to be common in patients with FMF. It was reported that the strikingly high gene frequencies for FMF in Mediterranean populations could be explained by the hypothesis of heterozygote selection. Heterozygotes might have a survival advantage by exhibiting greater but controlled inflammatory responses to a specific pathogen or class of pathogens. Although these enhanced inflammatory responses could be useful in the eradication of the pathogens, they might trigger the inflammation cascade resulting in non-suppurative inflammatory diseases in some genetically susceptible individuals [33]. To our knowledge, most of the patients diagnosed with both MS and FMF have been reported from Turkey [34,35]. A prospective research of MS in a small group of 17 FMF cases remained negative [17]. In 2006, Akman-Demir et al. reported that among 2268 cases with definite MS, there were 9 patients with coexisting FMF and three more patients had a neurological picture suggesting an unclassifiable demyelinating disorder [11]. All the reported FMF patients were referred to a Neurology department because of symptoms and/or signs of demyelinating CNS disease. The results of our study show that MEFV gene mutations were over-represented in the MS group. However, because of the limited number of the patients in our study, it is difficult to make a general conclusion that MEFV mutations are a risk factor for MS, or that MEFV gene may be a susceptibility gene to develop MS. Further investigations with larger study populations are necessary to confirm our results. In conclusion, the results of this study support the hypothesis that MS patients with MEFV mutation seem to be susceptible to develop a more progressive disease. Moreover, these data suggest that MEFV mutations may increase the risk of MS development. These and the proposed mode of action suggest a modifier role for pyrin in MS and in a wide spectrum of autoinflammatory and autoimmune diseases. Studies concerning the relationship between autoinflammatory
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Journal of the Neurological Sciences j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j n s
Evaluation of common mutations in the Mediterranean fever gene in Multiple Sclerosis patients: Is it a susceptibility gene? Aysun Unal a,⁎, Ahmet Dursun b, Ufuk Emre a, Nida F. Tascilar a, Handan Ankarali c a b c
Zonguldak Karaelmas University, Faculty of Medicine, Department of Neurology 67700, Kozlu, Zonguldak, Turkey Zonguldak Karaelmas University, Faculty of Medicine, Department of Medical Genetics 67700, Kozlu, Zonguldak, Turkey Zonguldak Karaelmas University, Faculty of Medicine, Department of Bioistatistics 67700, Kozlu, Zonguldak, Turkey
a r t i c l e
i n f o
Article history: Received 17 December 2008 Received in revised form 13 March 2010 Accepted 20 April 2010 Available online 18 May 2010 Keywords: Familial Mediterranean fever MEFV gene Pyrin domain Multiple Sclerosis Susceptibility Inflammation
a b s t r a c t Purpose: Multiple Sclerosis (MS) is a disease of the central nervous system characterized by multiple areas of inflammation and demyelination in the white matter of the brain and spinal cord. MEFV gene, which is the main factor in familial Mediterranean fever, is an intracellular regulator of inflammation. This study was designed to determine if known mutations in pyrin domain of MEFV gene are involved in MS and associated with MS morbidity. Methods: Fifty-three patients with MS and 66 healthy subjects, who were all Turkish, were included in this study. Five pyrin gene mutations (E148Q, M680I, M694V, M694I and V726A) were detected in the patients and controls by using the PRONTO™ FMF Basic Kit according to the manufacturer's instructions. Results: Pyrin gene mutations were found in 20 of the 53 MS patients (38%) and in seven of the 66 healthy subjects (11%). The frequency of total pyrin domain mutations was significantly higher in the MS patients than in the healthy subjects (p b 0.0001). The frequencies of M694V, E148Q and V726A mutations were significantly higher in the patients than in the healthy subjects (p = 0.02, p = 0.013, p = 0.004 respectively). The mean time to reach EDSS score 3.0 was earlier in the patients with MEFV gene mutation (p = 0.02) and the relapse rate was slightly higher among the MS patients carrying MEFV gene mutation (p = 0.04). Conclusion: The results of this study supported the hypothesis that MS patients with MEFV mutation seem to have the susceptibility to develop a more progressive disease. Moreover, these data suggest that MEFV mutations may increase the risk of MS development. © 2010 Elsevier B.V. All rights reserved.
1. Introduction Familial Mediterranean fever (FMF) is a recessive disorder characterized by episodes of fever with serositis or synovitis. The FMF gene (MEFV) was identified in 1997 by positional cloning [1]. According to INFEVERS (http://fmf.igh.cnrs.fr/infevers/) many MEFV mutations have been identified so far [2]. Five founder mutations V726A, M694V, M694I, M680I and E148Q account for 74% of FMF chromosomes from typical cases (Armenians, Arabs, Jews, and Turks) [3]. MEFV gene locus has been mapped to chromosome 16p 13.3 [1,4]. This gene encodes a 781-amino-acid protein named as pyrin. Pyrin is predominantly expressed in polymorphonuclear leukocytes (PMNs) and cytokine-activated monocytes [4,5]. Pyrin consists of four functional domains, a B-box zinc-finger domain, a coiled coil domain, a C-terminal B30.2 domain, and a 92-amino-acid N-terminal pyrin domain that is shared by a number of other proteins involved in apoptosis and inflammation [6]. The pyrin domain is a member of the
⁎ Corresponding author. Tel.: +90 5364428655; fax: +90 3722610155. E-mail address: [email protected] (A. Unal). 0022-510X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.jns.2010.04.008
six-helix bundle, death-domain superfamily that includes death domains (DD), death effector domains (DED), and caspase recruitment domains (CARDs) [7,8]. Thus, in patients with lack of pyrin protein (or its activity) due to hereditary defects, there is no suppression or inhibition of the inflammatory process, thereby leading to a full-blown attack [9]. Although FMF is a multisystem disease, its relationship with CNS involvement is not certain. Neurological complications during the course of FMF such as aseptic meningitis, pseudotumor cerebri, and nonspecific EEG abnormalities during febrile attacks have been previously reported [10,11]. Polyarteritis nodosa, microscopic polyangiitis and Henoch–Schonlein purpura are the three vasculitic diseases that have been known to be related with FMF. Diseases that have been proposed to be associated with FMF include Behcet's Disease (BD), inflammatory bowel disease and juvenile chronic arthritis [12–14]. These related diseases may also cause neurological complications. CNS demyelinating lesions in FMF patients have been reported rarely. Multiple Sclerosis (MS) is a pathogenically complex disease associated with a profound heterogeneity in the structural and immunopathologic features of the lesions, which vary depending on
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the stage of demyelinating activity or phase of the disease (early or late). In vitro and in vivo data indicate that multiple immune effector mechanisms can potentially lead to inflammation, focal demyelination, and tissue injury in MS [15,16]. A prospective search of MS in a small cohort of FMF cases remained negative [17]. In a previous study of a Jewish cohort with MS increasing disability occurred more rapidly in patients carrying the M694V allele of MEFV [18]. The pathophysiological relation between FMF and MS is still in debate. When combined with a Th1 related autoimmune disease, carrying a MEFV gene mutation may aggravate the disease course, as shown for carriers among patients with rheumatoid arthritis [19]. Also of note is the finding that healthy heterozygotes for MEFV mutations appear to have higher than normal blood levels of acute phase reactants, supporting the role of MEFV mutation in the induction of inflammation [20]. In this study, we aimed to determine whether mutations of pyrin domain of MEFV gene in Turkish MS patients are more prevalent than healthy controls and if they have an impact on the clinical disease course of MS. 2. Materials and methods Fifty-three MS patients and 66 healthy subjects were included in this study. The MS patients were followed-up on a regular basis in every 3–6 months at the Neurology outpatient-clinic of Zonguldak Karaelmas University, Faculty of Medicine. Informed consent was obtained from all the patients that participated in this study, and the local ethics committee of Zonguldak Karaelmas University, Faculty of Medicine approved the study. The Kurtzke Expanded Disease Status Scale (EDSS) scores of the patients in the follow-up examinations and all the clinical and demographic data of the patients were retrospectively obtained from the hospital computer database. The Progression index (EDSS/ duration of disease [years]) was determined in each patient included in the study. Blood samples of the MS patients were collected at the Medical Genetics Department of Zonguldak Karaelmas University, Faculty of Medicine. Genomic DNA was extracted from the peripheral blood leucocytes using the E.Z.N.A® Blood DNA Isolation Kit (Cat. No: D3392-02) according to the manufacturer's instructions. Five pyrin gene mutations (E148Q, M680I, M694V, M694I and V726A) were determined in the patients and controls by using the PRONTO™ FMF
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Basic Kit (Sayvon Diagnostic Ltd. Cat. No. 9904) according to the manufacturer's instructions. Descriptive statistics were computed as Mean ± SD and frequencies (count and percent). Prevalence of different MEFV gene mutations in the MS patients and healthy subjects was compared by the Fisher's exact chi-Square test. Pearson chi-square test was used to analyze the relation between categorical clinical and/or prognostic features of mutation carriers and non-carriers. Student t-test was preferred to analyze differences between the two groups about continuous clinical and/or prognostic features. Kaplan–Meier curve was drawn to present the probability of MS patients to reach EDSS: 3.0 or 6.0 during the disease course in relation to the MEFV genotype. p b 0.05 was considered statistically significant. SPSS (ver. 11.0) program was used for all the statistical analysis.
3. Results Between 2002 and 2008, a total of 90 cases with demyelinating disorders were registered in our clinic. Among these, 64 had definite MS; 16 had possible MS (mostly clinically isolated syndromes), and the remaining had other demyelinating diseases (acute disseminated encephalomyelitis, Devic's disease, recurrent optic neuritis, tumorlike or other unclassified demyelinating disorders). Fifty-three definite MS patients fulfilling Mc Donald's criteria were included in this study [21]. Eleven definite MS patients were excluded due to inadequate follow-up data. Thirty-six of the included 53 patients were female, and the mean age of the patients was 37 years. The mean follow-up time was 2.8 ± 1.7 years. None of these patients had clinical findings compatible with a systemic vasculitis. Screening of all the patients for systemic findings suggestive of BD was negative. Serologic screening tests for collagen vascular diseases (anti nuclear antibodies, antibodies against extractable nuclear antigens, antineutrophilic cytoplasmic antibodies, antiphospholipid antibodies and tests for syphilis) were all negative. One MS patient fulfilled modified Tel Hashomer criteria for diagnosis of FMF [22]. Fifty-two of the MS patients were not symptomatic for FMF and except one patient, none of the patients had a family history of FMF. The controls (n = 66) were all healthy subjects and had no symptoms for either FMF or MS. Fourteen of the controls were female, and the mean age of the controls was 34 years.
Table 1 Neurological and laboratory characteristics of MS patients carrying MEFV gene mutations (F: female, M: male, ON: optic neuritis, TM: transverse-myelitis, BS: brainstem, C: cerebellar, RR: relapsing–remitting, SP: secondary progressive, PP: primary progressive, PI: progression index, N: normal, ND: not determined, OB: oligoclonal band). Case no/sex
MEFV gene mutations
Age at onset (years)
Onset symptom
Disease duration (years)
Course
Outcome (final visit)
OB
1/F 2/F 3/F (Fig. 1a) 4/F (Fig. 1g–h) 5/M 6/M 7/F (Fig. 1f) 8/F 9/F (Fig. 1c) 10/F (Fig. 1e) 11/F 12/F 13/F 14/F 15/M 16/M 17/F (Fig. 1d) 18/F 19/M 20/F (Fig. 1b)
M694V /M694V V726A/V726A M694V/– V726A/– E148Q/– E148Q/– M694V/V726A E148Q/– E148Q/– M694V/– M694V/– M694I/– E148Q/– M694V/– M694V/– E148Q/– M694V/– V726A/– M680I 694V/E148Q
40 28 30 39 32 49 20 30 34 27 23 29 35 33 38 37 22 33 47 45
ON TM BS ON BS Pyramidal BS ON Pyramidal BS ON ON Sensorial ON Pyramidal BS BS ON BS Pyramidal
6 10 3 5 2 8 3 9 8 4 12 20 8 2 10 1 4 7 2 7
SP SP SP RR RR SP RR RP RR RR RR SP RR RR RP RR RR PP RR SP
EDSS:6.5 PI: 1.08 EDSS: 7.0 PI:0.70 EDSS:6.0 PI:2.00 EDSS:3.0 PI:0.38 EDSS:2.0 PI:0.38 EDSS:3.0 PI:1.07 EDSS:1.0 PI:0.57 EDSS:3.5 PI:0.60 EDSS:4.0 PI:1.0 EDSS:1.0 PI:0.33 EDSS:1.0 PI:0.50 EDSS:7.5 PI:0.25 EDSS:1.5 PI: 0.08 EDSS:1.0 PI:0.19 EDSS:2.5 PI:0.50 EDSS:1.0 PI:1.0 EDSS:1.0 PI:0.25 EDSS:4.0 PI:0.50 EDSS:1.0 PI:0.39 EDSS:7.5 PI:0.25
(+) (+) (+) (+) (−) ND (+) (+) (+) (+) ND (+) ND (+) (−) (+) (−) ND (−) (+)
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Table 2 Frequency of common MEFV mutations among MS patients and healthy subjects. Mutation type
MS patients n/2n
Healthy subjects n/2n
p value
M694V E148Q M680I V726A Total
11/106 7/106 1/106 5/106 24/106
4/132 1/132 2/132 0/132 7/132
0.02 0.013 0.691 0.004 b0.0001
Pyrin gene mutations were found in 20 of the 53 MS patients (38%) (fifteen female and five male) (Table 1). One patient was homozygous for M694V and one patient was homozygous for V726A, whereas two patients were carrying two different mutations (M694V/V726A; M694V/E148Q). M694V mutation was recorded in ten; E148Q in seven, V726A in four, and 680I in one case. There was no patient with M694I mutation. In the control group, MEFV mutations were found in seven of the 66 healthy subjects (11%). Four of 66 (6%) controls had M694V mutation, one (2%) had E148Q mutation and two (3%) had M680I mutation. The frequency of total pyrin gene mutations was significantly higher in the MS patients than in the healthy subjects (p b 0.0001). The frequency of M694V, E148Q and V726A mutations was found significantly higher from healthy subjects (p = 0.02, p = 0.013, p = 0.004 respectively). The frequency of the M680I mutation was not significant (Table 2). In order to determine whether the presence of MEFV gene mutation could aggravate the clinical expression of MS, we compared the demographic and clinical variables of mutation carriers and noncarriers. No significant differences were found between the two groups for mean age, average disease duration, and the mean age at onset. The course of disease was Relapsing–Remitting (RR) in 11 (55%), Relapsing Progressive (RP) in 2 (10%), Secondary Progressive (SP) in 6 (30%) and primary progressive (PP) in one patient (5%) in carriers. RR course was seen in 23 (69.7%); RP course, in 2 (6.1%); SP course, in 7 (21.2%) and PP course in one patient in non-carrier group. No statistically significant differences were found between the two groups for disease course (p = 0.754) and symptoms of MS onset (p = 0.381). Mean EDSS score (in the last neurological examination) was 3.2 ± 2.4 in carriers and 2.1 ± 2.1 in non-carriers (median scores 2.75 and 1.5 respectively). There were five patients (25%) in carriers and three
Table 3 Clinical and prognostic features of MS patients carrying a common MEFV gene mutation (ON: optic neuritis, TM: transverse-myelitis, BS: brainstem symptoms, RR: relapsing– remitting, SP: secondary progressive, PP: primary progressive, ⁎:significant p-value).
Age Disease duration (years) Age at onset Disease course
Disease onset symptom
RR RP SP PP ON TM BS Pyramidal Sensorial Polysymp. Cognitive
Patients reaching EDSS: 3 Patients reaching EDSS: 6 Mean time to reach EDSS 3 (years) Mean time to reach EDSS 6 (years) Relapse rate in RRMS patients
Carriers
Non-carriers
p value
40 ± 8.5 6.6 ± 4.5 33.6 ± 7.9 11 (55%) 2 (10%) 6 (30%) 1 (5%) 7 (35%) 1 (5%) 7 (35%) 4 (20%) 1 (5%) – – n = 10 n=5 3.9 ± 1.6 6.2 ± 5.0 1.6 ± 1.1
36 ± 9.2 7.1 ± 5.9 29.1 ± 9.4 23 (69.7%) 2 (6.1%) 7 (21.1%) 1 (3%) 7 (21.1%) 7 (21.1%) 6 (18.2%) 7 (21.1%) 4 (12.1%) 1 (3%) 1 (3%) n = 12 n=3 7.6 ± 4.3 11.7 ± 9.1 1.1 ± 0.9
p = 0.133 p = 0.711 p = 0.062 p = 0.754
p = 0.381
p = 0.329 p = 0.137 p = 0.02* p = 0.303 p = 0.04*
patients (9.1%) in non-carriers whose EDSS scores were ≥6.0. The mean progression index was 0.6 ± 0.4 in the carriers and 0.4 ± 0.4 in non-carriers. In the carrier group, the rate of the patients with progression index ≥ 0.5 was 55% (n = 11) and in the non-carrier group, 24% (n = 8). Although the mean progression index scores of the carriers seem to be higher, the difference was not statistically significant (p = 0.103). The comparison of the mean times to reach EDSS score 3.0 showed that it was earlier in the patients with MEFV gene mutation (p = 0.02). The mean time to reach EDSS = 3.0 was 4 years in the carriers and 7.5 years in the non-carriers. The mean time to reach EDSS = 6.0 was 5 years vs 13.5 years, respectively. No significant differences were determined between the two groups for the time required to reach EDSS score 6.0 (Table 3). The statistical analysis performed after the exclusion of primary progressive cases revealed no differences between the carriers and non-carriers for age, disease duration, progression index, frequency of the patients with EDSS ≥ 3.0, and the mean time to reach EDSS = 3.0. There were no patients with EDSS ≥ 6.0. The only statistically significant result was that the annual relapse rate was slightly higher among MS patients carrying MEFV gene mutation (p = 0.044). MEFV 694V was the most frequent mutation type in our study; therefore, we compared the mean times to reach EDSS: 3.0 and 6.0 of M694V mutation carriers with the other mutation type carriers (E148Q or M680I or V726A) and non-carriers. We found no differences between the progression milestones of the three groups. In all patients Magnetic Resonance Imaging (MRI) scans revealed typical MS lesions fulfilling the MRI criteria suggested by Mc Donald et al. [21]. Demyelinating lesions with nodular or ring-like enhancement (Fig. 1) were present in ten of the 20 MEFV gene mutation carrier patients. Spinal cord lesions were detected in fourteen of the carriers. Sixteen of the patients with MEFV gene mutation were subjected to CSF examination. The CSF samples were acellular with normal protein and glucose levels. Oligoclonal IgG bands were detected in twelve of the sixteen patients (Table 1). 4. Discussion In the present study, MS patients showed an increased frequency of MEFV (M694V, E148Q V726A and M680I) mutations compared to unaffected ethnically matched controls (38% and 11%). The frequency of M694V, E148Q and V726 mutations individually was also found to be significantly higher (Table 2). Shinar et al. studied mutations in the MEFV gene in MS patients in 2003 in Israel. They reported that the combined frequency of three founder mutations M694V, E148Q and V726A, and each mutation alone showed no increased frequency in MS patients. In their statistical analysis, the mutations in their study seemed to have affected the disability and relapse rate of the disease in the non-Ashkenazi MS patients group [18]. When we used Kaplan– Meier curve analysis, we also found that mutation carriers showed a statistically significant decrease in the mean time to reach EDSS: 3.0 compared to non-carriers. The mean time to reach EDSS: 6.0 also seemed to be earlier in the carriers, but it was not statistically significant. In accordance with the results of the study by Shinar et al., the annual relapse rate was slightly higher among RRMS patients carrying MEFV gene mutation. To date, numerous studies evaluated the degree of correlation between genotype and phenotype among patients with FMF. These studies, comparing disease phenotype with MEFV genotype, have indicated that some mutations are more pathogenic than others, but that clinical expression of the disease can vary widely among patients with the same mutations [23,24]. In the populations most typically affected by FMF, the mutation that appears to disrupt the function of pyrin to the greatest extend encodes the M694V variant [25,26]. M694V mutation is not only associated with a more severe clinical picture of FMF, but also confers a higher risk for these patients developing AA amyloidosis. In our study group, two MS patients were
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Fig. 1. a) Axial flair MR imaging of patient 3 with periventricular ovoid hyperintense lesions b) T1 axial MR image of patient 20 with hypointense periventricular lesions (black-holes) and brain atrophy c) Sagittal T1 MR imaging of patient 9 with ring and nodular gadalinum enhancing periventricular lesions d) Axial T1 MR imaging of patient 17 with open-ring, ring and nodular gadalinum enhancing lesions e) Coronal T2 brainstem MR imaging of patient 10 with hyperintense lesions in pons, medulla-oblongata and spinal cord f) Axial T2 MR imaging of patient 7 with ponto-cerebellar hyperintense lesions g) Sagittal T2 cervical spinal MR imaging of patient 4 with C6 localized hyperintense lesion h) same lesion on axial T1 MR imaging with gadalinium enhancement.
homozygous for MEFV gene mutation. Of these patients, one patient was homozygous for M694V, and one was homozygous for V726A. The first one with diagnosis of secondary progressive MS, fulfilled modified Tel Hashomer criteria for diagnosis of FMF, and she responded well to immunosuppressive therapy with colchicum. She was operated three times with the diagnosis of appendicitis, inguinal hernia, and over torsion because of recurrent abdominal pain in her twenties. The family history of the patient revealed abdominal pain attacks in her father; who died at the age of 43 with no diagnosis. The patient also described skin lesions and arthritis in her wrists and knees resolving in a week for several years [27]. Furthermore, some individuals may have paired pathogenic MEFV mutations but do not have clinical features of FMF [3]. The case of the second MS patient with homozygous MEFV V726A mutation supports studies suggesting that V726A homozygotes have milder forms [28]. She had no clinical finding that may be associated with FMF. She presented with a secondary progressive MS with no response to immunomodulatory therapy and mitoxantrone. Shinar et al., in their study on MS patients, have reported that progression to disability was earlier in carriers of the M694V mutation [18]. In this study, we also found that MEFV 694V mutation was the most frequent type; however, there were no differences between the progression milestones of the different mutation carriers. The diagnosis of FMF still remains predominantly clinical since mutations are not always penetrant and cannot always be identified on both alleles [29]. Paired MEFV mutations were detected in 84% of FMF patients and single mutations in 12%. Mutations in the MEFV gene may also be involved in non FMF disorders [28,30,31]. Recent studies from Turkey reported the frequency of MEFV mutation carriers as 10%–25% in Turkish population [14,24,32]. We found the frequency of MEFV mutations in 11% of healthy individuals, and most of the mutation carrier MS patients were heterozygous. The genetic defect in FMF was proposed to be the impaired control of inflammation in response to certain recognized or unrecognized
stimuli. Indeed chronic inflammatory disorders such as vasculitidis and Behcet's disease were reported to be common in patients with FMF. It was reported that the strikingly high gene frequencies for FMF in Mediterranean populations could be explained by the hypothesis of heterozygote selection. Heterozygotes might have a survival advantage by exhibiting greater but controlled inflammatory responses to a specific pathogen or class of pathogens. Although these enhanced inflammatory responses could be useful in the eradication of the pathogens, they might trigger the inflammation cascade resulting in non-suppurative inflammatory diseases in some genetically susceptible individuals [33]. To our knowledge, most of the patients diagnosed with both MS and FMF have been reported from Turkey [34,35]. A prospective research of MS in a small group of 17 FMF cases remained negative [17]. In 2006, Akman-Demir et al. reported that among 2268 cases with definite MS, there were 9 patients with coexisting FMF and three more patients had a neurological picture suggesting an unclassifiable demyelinating disorder [11]. All the reported FMF patients were referred to a Neurology department because of symptoms and/or signs of demyelinating CNS disease. The results of our study show that MEFV gene mutations were over-represented in the MS group. However, because of the limited number of the patients in our study, it is difficult to make a general conclusion that MEFV mutations are a risk factor for MS, or that MEFV gene may be a susceptibility gene to develop MS. Further investigations with larger study populations are necessary to confirm our results. In conclusion, the results of this study support the hypothesis that MS patients with MEFV mutation seem to be susceptible to develop a more progressive disease. Moreover, these data suggest that MEFV mutations may increase the risk of MS development. These and the proposed mode of action suggest a modifier role for pyrin in MS and in a wide spectrum of autoinflammatory and autoimmune diseases. Studies concerning the relationship between autoinflammatory
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