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MEFV gene mutations in Egyptian children with familial Mediterranean fever: clinical versus genetic diagnosis.

Introduction

Familial Mediterranean fever (FMF) is an autosomal recessive disease characterized by recurrent self-limited attacks of fever associated with serositis which usually involves the peritoneum, pleura, or synovium. The disease affects mainly populations of the Mediterranean basin, especially Sephardic Jews, Turks, Armenians, and Arabs [1,2] .Yet, it is observed worldwide due to the widespread inter-continental travel in the twentieth century [3].

FMF is caused by mutations in the MEFV gene which encodes the anti-inflammatory pyrin protein. Pyrin protein is expressed mainly in neutrophils and it normally inhibits chemotactic factor (C5a) and interleukins 1b and 8. Mutations in the MEFV gene lead to autoinflammation and an exaggerated inflammatory response by reducing pyrin activity [4].

Due to the clinical heterogeneity of FMF and the lack of specific diagnostic biochemical tests, molecular genetic analysis may help establish a definitive diagnosis. This allows early implementation of prophylactic therapy with Colchicine that can substantially reduce the severity of the attacks and the risk of renal amyloidosis, which is the most serious complication of the disease [5].

Several Mediterranean populations have been studied extensively for MEFV mutations, whereas data on Egyptian patients are scarce [6,7]. The present study was conducted to identify and determine the frequencies of MEFV gene mutations in a cohort of Egyptian patients in whom FMF was diagnosed and to explore the presence of a possible correlation between the diverse genotypes and the phenotypic expressions of the disease.

Materials and Methods

This cross sectional study was conducted in the Rheumatology clinic, New Children Hospital, Cairo University in the period from February 2010 to February 2012. It included 61 patients with the clinical diagnosis of FMF. A clinical diagnosis of FMF was made according to published criteria of Simon et al. [8]. Major criteria were recurrent febrile episodes accompanied by peritonitis, synovitis, or pleuritic, Amyloidosis of AA-type without a predisposing disease and a favorable response to continuous Colchicine treatment. On the other hand, minor criteria were: recurrent febrile episodes, erysipelas-like erythema, and FMF in a first-degree relative. Definite diagnosis was based on the presence of two major criteria or one major and two minor criteria. Probable diagnosis of FMF requires one major and one minor criteria.

All patients enrolled in the study were submitted to a full history regarding their age, sex, age of onset of inflammatory attacks, and the frequency and duration of these attacks. Any history of renal affection including amyloidosis was also recorded. It also included a history of Colchicine intake and the response to therapy. Detailed family history was obtained from each patient with special emphasis on parental consanguinity, and family history of FMF symptoms and chronic renal failure. Then, all patients underwent laboratory investigations including complete blood count ESR, urinalysis and mutation analysis.

Mutation analysis:

Genomic DNA was extracted from the whole blood according to the standard procedures using QIAamp Spin Columns by QIAamp DNA Blood Kits [Cat.No.51104]. Each DNA sample was tested for the presence of the 3 commonest mutations: M680I, V726A and M694V. Mutations were assessed by amplifying genomic DNA with use of primers [9].

M6801, V726A and M694V mutations were analyzed by amplification refractory mutation system (ARMS). The MEFV region covering the three mutations was amplified using primers [10].

Statistical methods:

Data was analyzed using SPSSwin statistical package version 12; data was summarized as mean, standard deviation (SD), standard error (SE) and percentage (%).Non-parametric test (Mann-Whitney U test) was used for analysis of 2 independent quantitative variables. Chi Square test was used for analysis of qualitative data. P-value is consider significant if [less than or equal to] 0.05.

Results:

The 17 patients with positive mutations included 9 females (52.9%) and 8 males (47.1%) with F: M = 1.13: 1. Their ages ranged from 2.0 to 13.0 years (mean [+ or -] SD = 7.8 [+ or -] 2.8). The age of onset of FMF ranged from 1.0 to 12.0 years (mean [+ or -] SD = 5.1 [+ or -] 3.1). They had variable duration of FMF attacks ranged from 1.0 to 7.0 days (mean [+ or -] SD = 2.5 [+ or -] 1.5). The duration of FMF ranged from 1.0 to 7.0 years (mean [+ or -] SD = 2.7 [+ or -] 2.1)

According to the result of gene mutation, the FMF patients are classified into 2 groups; Group I included patients with positive gene mutations (i.e. at least one gene mutation was positive) (17 patients = 27.9%) and group II which included patients with negative gene mutations (44 patients = 72.1%)

The age was significantly lower in patients with positive mutations while the duration of FMF was more prolonged in patients with negative mutations compared to patients with positive mutation.

Furthermore, the attacks were significantly more frequent in patients with positive mutations compared to patients with negative mutations

To study the association between various MEFV gene mutations and the clinical severity of FMF. We compared between FMF patients with positive M680I only (N=5) and FMF patients with positive V726A only (N=10) regarding their clinical and laboratory parameters; figure 1 and figure 2.

Discussion:

Clinical diagnostic criteria of FMF can lead to many patients being undiagnosed, especially those with atypical presentations or with a mild disease. Adoption of a molecular diagnostic approach can result in a better definitive diagnosis. In this study, we determined the frequencies of the three commonest mutations in the MEFV gene (M694V, M680I and V726A) in 61 Egyptian patients with FMF. Positive mutations were found in 17 patients (27.87%) which is less than that reported by Mattit et al. [11] (89%), who screened for 5 genes mutations (M694V, V726A, M694I, M680I and E148Q), and Kinikli et al. [12] (92.5%) whose performed strategy included the screening of almost all mutations known to date. It has been suggested that the proportion of unidentified mutations is higher in Arab patients with FMF than in other affected ethnic groups [13,14]. The present study explored only 17 mutations; therefore, it is possible that patients in whom no mutations or a single mutant allele was detected may carry other known mutations that were not screened in this study or other previously unidentified mutations in the MEFV gene.

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

No significant differences were observed between the 2 groups (p>0.05) for all the studied laboratory parameters

In our study, V726A gene mutation was the most frequent mutation (19.7%), followed by M6801 mutation (11.5%), and M694V mutation was reported in one patient (1.6%). Similar results were reported by Brik et al. (15) in Arabs, Druze and Iraqi Jews who bear mostly the V726A mutation, also Majeed et al. [16] reported that V726A was the prevalent mutation among Arab patients with FMF.

On the contrary, Settin et al. [6] reported M694V to be the most frequent mutation in Egyptian pediatric patients with FMF who are living in the Nile Delta region. The discrepancies in mutation frequencies between our study and that of Settin et al. may be explained by the smaller number of subjects in their study, which included 42 patients with MEFV gene mutations, 21 of whom carried two mutant alleles and the remaining 21 carried only one mutant allele. In addition, 92.4% of their patients presented at an age below 10 years compared to the patients of our study whose age ranged between 2 and 16 years. This may account for the high prevalence of M694V in their patients, which is generally considered a severe mutation associated with an early onset of symptoms.

In our study, there was a significant difference between the group of positive mutations and the group of negative mutations, regarding the age of the patients at the time of the study, being younger in patients with positive gene mutations compared to patients with negative mutations, and also regarding the duration of FMF attacks being more prolonged in patients with negative mutations. On the other hand, Brik et al. [15] found no association between age of FMF onset and the presence of a FMF gene mutation. Conversely, Seyahi et al. [17] found that patients with FMF mutations were significantly older, had a more prolonged duration of FMF. In addition, the current study revealed that, in patients with positive mutations the attacks of FMF were significantly more frequent compared to patients with negative mutations, this result was similar to that reported by several authors [17, 18].

In present study, no significant relation between FMF gene mutation and gender was detected; these results were in agreement with the data of George [19], and Mijatovic et al. [20]. On the other hand, Majeed et al.[16] found a slight female preponderance with a female: male ratio of 54%, however, other authors found FMF gene mutation to be more frequent in males (P<0.05) [ 21,22]. They explained this male preponderance by the greater resistance of females to pain and the possibility of confusing abdominal with gynecological pain.

Several authors studied the association between various MEFV gene mutations and the clinical severity of FMF [23, 24], however the reported data were inconsistent. Some investigators reported an association between the M694V, especially in the homozygous state, and the severity of FMF and the development of amyloidosis [25,26], whereas others failed to find evidence for such association [27,28].

In the present study, NO SIGNIFICANT differences were observed in both the clinical and laboratory data in FMF patients with positive M680I (N= 5) when compared to those with positive V726A only (N=10) except the positive family history which was more frequent in patients with V726A mutation. These results were similar to that of Onen et al [29] but disagree with those of, Gershoni-Baruch et al. [21] and ; Medlej-Hashim [22] who reported a significant correlation between FMF patients genotypes with various phenotypes of the disease especially amyloidosis which was common with M694V mutation.

There has been a considerable disagreement among various investigators on the incidence of secondary amyloidosis complicating FMF. Several authors found no specific genotype linked with the development of amyloidosis [27,30] while other recent studies reported amyloidosis being more frequent in patients with homozygous M694V mutations [2,3]. Furthermore, It has been noted that amyloidosis is less common in Arab patients with FMF and that the disease seems to run a milder course in Arabs than in other ethnic groups [32, 33]. In the present study, one patient developed amyloidosis, and the gene study revealed positive V726A gene mutation.

Conclusion:

This study allowed us to clearly establish the spectrum for MEFV common mutations among Egyptian FMF patients. Our results proved that there is no consistency in the association between specific MEFV mutations and phenotypic features. The present data also indicated that specific FMF mutations are not the sole determinants of the disease severity nor for the development of amyloidosis. Therefore, the presence of phenotypic variations of the disease and the development of amyloidosis can be explained by unknown factors and/ or the presence of other genetic changes.

References

[1.] Ritis, K., S. Giaglis, N. Spathari, et al. 2004. Non-isotopic RNase cleavage assay for mutation detection in MEFV, the gene responsible for familial Mediterranean fever, in a cohort of Greek patients. Ann Rheum Dis; 63: 438-43.

[2.] Fragouli, E., E. Eliopoulos, E. Petraki, et al. 2008. Familial Mediterranean fever in Crete: a genetic and structural biological approach in a population of intermediate risk. Clin Genet., 73: 152-159.

[3.] Bakkaloglu, A., 2003. Familial Mediterranean fever. Pediatr Nephrol, 18: 853-9.

[4.] Stehlik, C., J.C. Reed., 2004. The PYRIN connection: novel players in innate immunity and inflammation. J Exp Med., 200: 551-558.

[5.] Ben-Chetrit, E., M. Levy, 1998. Colchicine: 1998 update. Semin Arthritis Rheum., 28: 48-59.

[6.] Settin, A., R. El-Baz, M. Abd Rasool, et al., 2007. Clinical and molecular diagnosis of familial Mediterranean fever in Egyptian children. J Gastrointest Liver Dis., 16: 141-145.

[7.] Al-Alami, J.R., M.K. Tayeh, D.A. Najib, et al. 2003. Familial Mediterranean fever mutation frequencies and carrier rates among a mixed Arabic population. Saudi Med., J 24: 1055-1059.

[8.] Simon, A., J. Van der Meer, R. Vesel, et al., 2004. Approach to genetic analysis in the diagnoses of hereditary autoinflammatory syndromes. Oxford J; 45(3): 269-73.

[9.] Eisenberg, S., I. Aksentijevich, Z. Deng, et al., 1998. Diagnosis of familial Mediterranean fever by a molecular genetics method. Ann of Int Med., 129(7): 539-42.

[10.] Brik, R., D. Litmanovitz, D. Berkowitz, et al., 2001. Incidence of familial Mediterranean fever among children of Mediterranean extraction with functional abdominal pain. J Peddiatr., 138: 759-62.

[11.] Mattit, H., M. Toma, S. Al-Cheikh, et al. 2006. Familial Mediterranean fever in Syrian population: gene mutation frequencies, carrier rates and phenotype- genotype correlation. Eur J Med Genet., 49(6): 481-6.

[12.] Kinikli, G., M. Bektas, M. Misirlioglu, et al. 2005. Relationship between HLA-DR, HLA-DQ alleles and MEFV gene mutation in familial Mediterranean fever (FMF) patient. Rhem., 16(3): 143-6.

[13.] Padeh, S., Y. Shinar, E. Pras, et al. 2003. Clinical and diagnostic value of genetic testing in 216 Israeli children with familial Mediterranean fever. J Rheumatol., 30: 185-190.

[14.] Giaglis, S., V. Papadopoulos, K. Kambas, et al. 2007. MEFV alterations and population genetics analysis in a large cohort of Greek patients with familial Mediterranean fever. Clin Genet., 71: 458-467.

[15.] Brik, R., D. Litmanovitz, D. Berkowitz, et al. 2001. Incidence of familial Mediterranean fever among children of Mediterranean extraction with functional abdominal pain. J Peddiatr., 138: 759-62.

[16.] Majeed, H., M. Rawashdeh, H. El-Shanti, et al. 1999. Familial Mediterranean fever in children: the expanded clinical profile. Oxford J; 92: 309-18.

[17.] Seyahi, E., H. Ozdogan, S. Celik, et al., 2006. Treatment options in colchicine resistant familial Mediterranean fever patients: Thalidomide and etanerecept as adjunctive agents. Clin Exp Rheumatol., 24(5): 99-103.

[18.] Atagunduz, M., S. Tuglular, G. Kantara, et al. 2004. Association of FMF related (MEFV) point mutations with secondary and FMF amyloidosis. Nephron Clin Pract., 96(4): c131-5.

[19.] George, E., 1998. Genetics of familial Mediterranean fever and its implication. Ann Intern Med., 129(7): 581-5.

[20.] Mijatovic, V., P.G. Hompes and M.G. Wouters, 2003. Familial Mediterranean fever and its implications for fertility and pregnancy. Eur. J. Obstet. Gynecol. Reprod. Biol., 108: 171-6.

[21.] Gershoni-Baruch, R., R. Brik, R. Lidar, et al. 2003. The contribution of genotypes at the MEFV and SAA1 loci to amyloidosis and disease severity in patients with familial Mediterranean fever. Arthritis Rheum., 48(4): 1149-55.

[22.] Medlej-Hashim, M., V. Delague, E. Chouery, et al. 2004. Amyloidosis in familial Mediterranean fever patients: Correlation with MEFV genotype and SAA1 and MICA polymorphism effects. BMC Med Genet., 10: 5-15.

[23.] Ozen, S., A. Bakkaloglu, E. Yilmaz, 2002. Pyrin Q148 mutation and familial Mediterranean fever. Q J Med., 95: 332-333.

[24.] Topaloglu, R., F. Ozaltin, E. Yilmaz, et al. 2005. E148Q is a disease causing MEFV mutation:a phenotypic evaluation in patients with familial Mediterranean fever. Ann Rheum Dis., 64: 750-752.

[25.] Padeh, S., Y. Shinar, E. Pras, et al. 2003. Clinical and diagnostic value of genetic testing in 216 Israeli children with familial Mediterranean fever. J Rheumatol., 30: 185-90.

[26.] Cazeneuve, C., T. Sarkisian, C. Pecheux, et al., 1999. MEFV-gene analysis in Armenian patients with familial Mediterranean fever: diagnostic value and unfavorable renal prognosis of the M694V homozygous: genotype genetic and therapeutic implications. Am J Hum Genet., 65: 88-97.

[27.] Yalcinkaya, F., N. Cakar, M. Misirhoglu, et al. 2000. Genotype-phenotype correlation in a large group of Turkish patients with familial Mediterranean fever: evidence for mutation-independent amyloidosis. Rheumatology, 39: 67-72.

[28.] Samuels, J., I. Aksentijevich, Y. Torosyan, et al., 1998. Familial Mediterranean Fever at the Mellenium. Clinical spectrum, ancient mutations and a survey of 100 American referrals to the National Institutes of Health. Medicine., 77: 268-97.

[29.] Onen, F., 2006. Familial Mediterranean fever. Rheumatol Int., 26(6): 489-96.

[30.] Orbach, H and E. Ben-Chetrit, 2001. Familial Mediterranean fever: a review and update. Rheum., 92(6): 421-30.

[31.] Chaabouni, H.B., M. Ksantini, R. Mrad, et al. 2007. MEFV mutations in Tunisian patients suffering from familial Mediterranean fever. Semin Arthritis Rheum., 36: 397-401.

[32.] Majeed, H.A., M. El-Khateeb, H. El-Shanti, et al. 2005. The spectrum of familial Mediterranean fever gene mutations in Arabs: report of a large series. Semin Arthritis Rheum., 34: 813-818.

[33.] Tunca, M., S. Akar, F. Onen, et al. 2005. Turkish FMF Study Group. familial Mediterranean fever (FMF) in Turkey: results of a nationwide multicenter study. Medicine., 84: 1-11.

Samia Salah, Mostafa Zakaria, Mona Azez, Hoda Marzouk

Department of Pediatrics, Cairo university, and department of clinical pathology, Cairo university

Corresponding Author

Mostafa zakaria MD, Cairo university, Cairo 28 Abu El hool street--Giza

E-mail: Drmostafazakaria@hotmail.com
Table 1: shows the demographic data of the study cases

Variables                 Number (61)   Percentage

Sex:
Males                     30            49.2
Females                   31            50.8

Parental consanguinity:
Positive                  12            19.7
Negative                  49            80.3

Family history of FMF:
Positive                  16            26.2
Negative                  45            73.8

Table 2: shows clinical data of the study cases

Variables                          Minimum   Maximum   Mean   SD (#)

Age (yr)                           2.0       16.0      9.5    3.6
Age of onset (yr)                  1.0       14.0      5.0    3.2
Duration of attacks (days)         1.0       15.0      3.1    3.1
Frequency of attacks (every/days)  10        90        32.6   23.7
Duration of disease (yr)           1.0       16.0      5.7    4.3

(#) SD : Standard deviation.

Table 3: shows the clinical presentations of the study cases

Variables                   Number(61)   Percentage

Typical attacks of fever:
Positive                    49           80.3
Negative                    12           19.7

Abdominal pain:
Positive                    54           88.5
Negative                    7            11.5

Chest pain:
Positive                    25           41
Negative                    36           59

Joint affection:
Positive                    41           67.2
Negative                    20           32.8

Skin affection:
Positive                    11           18
Negative                    50           82

Muscle pain:
Positive                    7            11.5
Negative                    54           88.5

Scrotal pain:
Positive                    1            1.6
Negative                    60           98.4
Vomiting:
Positive                    14           23
Negative                    47           77

Colchicine response:
Present                     58           95.1
Absent                      3            4.9
Amyloidosis:
Positive                    1            1.6
Negative                    60           98.4

Table 4: shows the frequency of mutations in the study cases

Variables    Number (61)   Percentage

V726A:
Positive     12            19.7
Negative     49            80.3

M680I:
Positive     7             11.5
Negative     54            88.5

M694V:
Positive     1             1.6
Negative     60            98.4

Laboratory screening of gene mutations in the study cases revealed

* 10 patients with V726A gene mutation "9 patients were
heterozygous and 1 patient was homozygous".

* 5 patients with M680I gene mutation "all
were heterozygous".

* 1 patient with compound heterozygous V726A and
M680I genes mutations.

* 1 patient with compound heterozygous V726A, M680I and
M694V genes mutations.

Table 5: shows the demographic data of the study cases with positive and
negative mutations

Variables        Positive                Negative              P-value
                 mutation                mutation

                 Number     Percentage   Number   Percentage
                 (17)                    (44)

Sex:

Males            8          47.1         22         50
Females          9          52.9         22         50         0.5

Parental
consanguinity:

Positive         1          5.9          11         25
Negative         16         94.1         33         75         0.09

Family history

Positive         7          41.2         9          20.5       0.09
Negative         10         58.8         35         79.5

Table 6: shows the clinical data of the study cases with positive
and negative mutations

Variables           Positive mutation   Negative mutation   P-value
                    Mean [+ or -] SE    Mean [+ or -] SE
                    N = 17              N = 44

Age (yr)            7.8 [+ or -] 0.7    10.2 [+ or -] 0.6   0.008
Age of onset (yr)   5.1 [+ or -] 0.7    5.0 [+ or -] 0.5    0.8
Duration of         2.5 [+ or -] 0.4    3.4 [+ or -] 0.5    0.8
  attacks (days)
Frequency of        23.9 [+ or -] 3.7   35.9 [+ or -] 3.9   0.05
  attacks
  (every/days)
Duration of the     2.7 [+ or -] 0.5    6.8 [+ or -] 0.7    0.001
  disease (yr)

Table 7: shows the clinical presentations of the study cases with
positive and negative mutations No significant differences were
observed between the 2 groups regarding the presenting symptoms

Variables          Positive mutation     Negative mutation    P-value
                      N = 17                N = 44

                  Number   Percentage   Number   Percentage

Typical attack
  of fever:
Positive          14       82.4         35       79.5
Negative          3        17.6         9        20.5         0.6
Abdominal pain:
Positive          14       82.4         40       90.9         0.3
Negative          3        17.6         4        9.1
Chest pain:
Positive          7        41.2         18       40.9         0.6
Negative          10       58.8         26       59.1
Joint
  affection:
Positive          11       64.7         30       68.2         0.5
Negative          6        35.3         14       31.8
Skin affection:
Positive          5        29.4         6        13.6         0.1
Negative          12       70.6         38       86.4
Muscle pain:
Positive          1        5.9          6        13.6         0.4
Negative          16       94.1         38       86.4
Scrotal pain:
Positive          1        5.9          0        0            0.3
Negative          16       94.1         44       100
Vomiting:
Positive          5        29.4         9        20.5         0.3
Negative          12       70.6         35       79.5
Colchicine
  response:
Present           16       94.1         42       95.5         0.6
Absent            1        5.9          2        4.5
Amyloidosis:
Positive          1        5.9          0        0            0.3
Negative          16       94.1         44       100

Table 8: shows the laboratory data of the study cases with positive
and negative mutations

Variables      Positive                Negative                P-value
               mutation                mutation
               N = 17                  N = 44

               Number     Percentage   Number     Percentage

WBCs:

High           9          52.9         31         70.5         0.2
Normal         8          47.1         13         29.5

ESR:

High           8          47.1         36         81.8         0.01 *
Normal         9          52.9         8          81.2

CRP:

Positive       10         58.8         21         47.7         0.3
Negative       7          41.2         23         52.3

Proteinuria:

Positive       1          5.9          0          0            0.3
Negative       16         94.1         44         100

ESR was significantly higher in patients with negative mutations (81.8%)
compared to patients with positive mutations (47.1) (p=0.01). No
significant differences were observed regarding the other studied
laboratory parameters
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Title Annotation:ORIGINAL ARTICLE
Author:Salah, Samia; Zakaria, Mostafa; Azez, Mona; Marzouk, Hoda
Publication:Advances in Environmental Biology
Article Type:Report
Geographic Code:7EGYP
Date:Oct 1, 2012
Words:3631
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