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A study of familial Mediterranean Fever (MEFV) gene mutations in Egyptian children with type 1 diabetes mellitus
European Journal of Medical Genetics 58 (2015) 31e34
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical research
A study of familial Mediterranean Fever (MEFV) gene mutations in Egyptian children with type 1 diabetes mellitus Ghada Mohammad Anwar a, Hanan M. Fouad b, *, Amal Abd El-Hamid b, Faten Mahmoud c, Noha Musa a, Hala Lotfi a, Nermine Salah a a b c
Pediatric Department, Cairo University, Cairo, Egypt Pediatric Department, National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt Chemical Department, National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 July 2014 Accepted 19 October 2014 Available online 4 November 2014
Background/Aims: An association of type 1 DM and familial Mediterranean fever (FMF) has been newly reported in the medical literature. The aim of the present work was to investigate frequency of MEFV gene mutations in Egyptian children with type 1 diabetes mellitus. Methods: Forty five children with type 1 DM were screened for Mediterranean Fever (MEFV) gene mutation. Forty one healthy control subjects were included. Identification of FMF gene mutation was done based on polymerase chain reaction (PCR) and reverse hybridization. The assay covers 12 mutations in the FMF gene: E148Q e P369S e F479L e M680I (G/C) e M680I (G/A) e I692del e M694V e M694I e K695ReV726A e A744S and R761H. Results: Among the screened diabetics, the overall frequency of MEFV gene mutations was 42.2% and among the control group it was 34.1% with no significant difference. Fourteen out of 45 diabetic children (31.1%) were heterozygous (E148Q in 7 children, A744S in 3 children, V726A in 2 children, M680I (G/C) in 1 child and P369S in1 child), while 5 children (11.1%) were compound heterozygous (M694V/M694I in 2 children, E148Q/K695R mutations in 1 child, E148Q/M694I in 1 child and E148Q/V726A in 1 child). The control group showed heterozygous mutation in 34.1% of cases (E148Q mutation in 14.6%, V726A in 12.2%, M680I (G/C) in 4.9% and M694V in 2.4%). Conclusion: No significant difference in mutation frequency between diabetic and non-diabetic children. We have high carrier rate of MEFV gene mutations among Egyptian population probably due to high consanguinity. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Type 1 diabetes Familial Mediterranean fever Children MEFV gene mutation
1. Introduction Familial Mediterranean fever (FMF) is an autoinflammatory diseases that is often associated with other autoimmune diseases such as ankylosing spondylitis, rheumatoid arthritis, polyarteritis nodosa (PAN), Behcet, multiple sclerosis (MS), and Systemic Lupus (SLE), Henoch-Schönlein purpura, and Hashimoto’s thyroiditis [Yildiz et al., 2010; Aksu and Keser, 2011; Erten et al., 2013; Ergul et al., 2013]. An association of type 1 DM and FMF has been newly reported in the medical literature [Atabek et al., 2006; Bas¸ et al., 2009]. FMF is an autosomal recessive disorder and it comprises two phenotypes: FMF type 1 is characterized by recurrent short
* Corresponding author. E-mail address: [email protected] (H.M. Fouad). http://dx.doi.org/10.1016/j.ejmg.2014.10.005 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
episodes of inflammation and serositis including fever, peritonitis, synovitis, pleuritis, and rarely, pericarditis and meningitis. FMF type 2 is characterized by amyloidosis as the first clinical manifestation of FMF in an otherwise asymptomatic individual [Shohat and Halpern, 2011]. In 1997 the gene responsible for FMF, MEFV, was identified [Ritis et al., 2004; FMF International Consortium, 1997]. FMF is due to recessive mutations in the MEFV (for Mediterranean Fever) gene, located on the short arm of the chromosome 16 (16p13.3), consisting of 10 exons and 781 codons [Pras et al., 1992]. The type and combination of the mutations define a severely or weakly expressed phenotype [Ben-Chetrit, 2003; Dodé et al., 2000; Gershoni-Baruch et al., 2002], but, on the other hand, subjects carrying two mutations may not express the disease [Kogan et al., 2001]. Mutations in the pyrin/marenostrin (MEFV) gene have been identified in the majority of FMF patients [Aksentijevich et al., 1999;
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G.M. Anwar et al. / European Journal of Medical Genetics 58 (2015) 31e34
Bernot et al., 1998]. 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 vast majority of FMF chromosomes identified [Aksentijevich et al., 1999; Bernot et al., 1998; Gershoni-Baruch et al., 2001; Kogan et al., 2001]. It has been established, that the phenotypic variability of the disease is, at least, partly due to allelic heterogeneity. Mutation M694V is associated with a severe phenotype and amyloidosis, and mutation V726A with a milder form of the disease [Cazeneuve et al., 1999; Dewalle et al., 1998]. The observation that V726A e E148Q allele is associated with a severe disease and strongly predisposes to renal amyloidosis, highlights the need to prescribe colchicine, as early as possible, to patients who carry the complex allele. Since amyloidosis, which is preferentially associated with either the M694V or the V726A e E148Q allele, may develop in FMF patients who do not have attacks of serositis, the need to screen asymptomatic family members, for mutations in the MEFV gene, is underlined [Gershoni-Baruch et al., 2002]. Up to our knowledge, there are no previous researches investigated the frequency of MEFV gene mutation in Egyptian type 1 diabetic patients. The aim of the present work was to investigate frequency of 12 MEFV gene mutations in Egyptian children with type 1 diabetes mellitus. 2. Subjects and methods Forty five children with type 1 DM, attending the Diabetes Endocrine and Metabolism Pediatric Unit at Cairo University Pediatric Hospital were selected randomly and screened for Mediterranean Fever (MEFV) gene mutation. Inclusion criteria were Diabetic children of any age and both sexes. Approval by the ethical committee was obtained. Enrollment was done after obtaining verbal consent from parents. Enrolled children were subjected to medical history (age, sex, consanguinity of parents, duration of diabetes, fever, abdominal pain, arthralgia, pleurisy, myalgia, rash), medical examination (for different systems; joints, chest, cardiovascular system, central nervous system, abdomin), routine laboratory investigations including HbA1c, thyroid profile, and urinary albumin/creatinine ratio were done in all patients and blood samples were obtained for assay of FMF gene mutation. Exclusion criteria were any patient who was already diagnosed as FMF or other rheumatic disorder. Forty one healthy; age and sex matched; children were included as a control group.
frequency and percentage. Chi-square test, unpaired t-student test were used to compare children with and those without MEFV gene mutation. Level of significance was P 0.05 for all statistical analyses. 3. Results Among the 45 diabetic children included in the study, 49% were male and 51% were females. The mean age of the children was 10.1 3.7 years (2.5e17), and the mean duration of diabetes was 3 1.7 years (1e8), mean HbA1c was 8.23 1.48, and the mean insulin dose was 1.19 0.28 U/Kg/day. Positive consanguinity of parents was detected in 44.4%. Overt nephropathy was detected in one patient, microalbuminuria in one patient, and hypothyroidism in one patient. Descriptive data of the diabetic group was summarized in Table 1. Among the control group, 44% were male and 56% were females and their ages ranged from 5 to 16 years. Positive consanguinity of parents was detected in 42%. Among the screened diabetic children, 19 (42.2%) were positive for MEFV gene mutations, while 26 (57.8%) were negative. Fourteen out of 45 children (31.1%) were heterozygous (E148Q in 7 children, A744S in 3 children, V726A in 2 children, M680I (G/C) in 1 child and P369S in1 child), while 5 children (11.1%) were compound heterozygous (M694V/M694I in 2 children, E148Q/K695R in 1 child, E148Q/M694I in 1 child and E148Q/V726A in 1 child) (Table 2). Among the screened control subjects, 14 (34%) were positive for MEFV gene mutations, while 27 (66%) were negative, with no significant difference between diabetic and control subjects (P ¼ 0.09). In the control group, all subjects were heterozygous (E148Q in 6 children, V726A in 5 children, M680I in 2 children, and M694V in one child). A comparison between diabetic and control subjects regarding MEFV gene mutations is shown in Table 2. Comparison of diabetic children with positive and negative MEFV gene mutation showed no significant difference in both groups regarding recurrent abdominal pain, arthralgia, nephropathy, microalbuminuria, hypothyroidism and hepatomegaly due to autoimmune hepatitis (Table 3). Comparison between patients with heterozygous mutation and compound heterozygous gene mutation was shown in Table 4. Overt nephropathy was found in one patient in compound heterozygous group (M694V/M694I). One patient with heterozygous MEFV gene mutation has family history of type 1 diabetes. Compound heterozygous mutation was detected in 2 patients with positive consanguinity as well as 3 patients with negative consanguinity, mutation types are shown in Table 5.
2.1. FMF gene mutation analysis A 5 ml EDTA blood sample was obtained by venipuncture for FMF assay. Identification of FMF gene mutation was done based on polymerase chain reaction (PCR) and reverse hybridization. Procedure includes three steps: DNA isolation, PCR amplification using biotinylated primers and hybridization of the amplification products to a test strip containing allele specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences are detected using streptavidin alkaline phosphatase and color substrate. The assay covers 12 mutations in the FMF gene: E148Q e P369S e F479L e M680I (G/C) e M680I (G/A) e I692del e M694V e M694I e K695ReV726A e A744S and R761H. 2.2. Statistical analysis Data were analyzed by Statistical Package for Social Science (SPSS) program version 17.0. Descriptive statistics such as mean (standard deviation) for normally distributed data were calculated for quantitative parameters. Qualitative data were summarized as
Table 1 Descriptive data of the diabetic group. Data Sex Male Female Residence Urban Rural Positive consanguinity History Abdominal pain Arthralgia Hepatomegaly Renal assessment Overt Nephropathy Microalbuminuria Associated conditions Hypothyroidism Autoimmune hepatitis
No (%) 22 (48.9) 23 (51.1) 44 (97.8) 1 (2.2) 20 (44.4) 24 (39.3) 20 (32.8) 1 (2.2) 1 (2.2) 1 (2.2) 1 (2.2) 1 (2.2)
G.M. Anwar et al. / European Journal of Medical Genetics 58 (2015) 31e34 Table 2 Comparison of MEFV gene mutation between diabetic and control subjects.
33
Table 5 Mutation types in consanguineous and non-consanguineous parents.
MEFV gene mutation
Diabetic children N ¼ 41
Controls N ¼ 41
P value
Consanguinity E148Q - K695R E148Q-V726A E148V-M694I M694V - M694I
Negative Heterozygous; E148Q A744S V726A M680I (G/C) P369S M694V Compound heterozygous; M694V/M694I E148Q/K695R E148Q/M694I E148Q/V726A
26 (57.8) 14 (31.1) 7 (15.6) 3 (6.7) 2 (4.4) 1 (2.2) 1 (2.2) 0 5 (11.1) 2 (4.4) 1 (2.2) 1 (2.2) 1 (2.2)
27 (65.9) 14 (34.1) 6 (14.6) 0 5 (12.2) 2 (4.9) 0 1 (2.4) 0
0.09
Negative Positive
Table 3 Comparison between diabetic children with positive and negative MEFV gene mutation. MEFV gene mutation
Age; Mean SD Duration of DM; Mean SD Sex; N (%) Male Female Residence; N (%) Urban Rural History; N (%) Abdominal pain Arthralgia Positive consanguinity; N (%) Renal assessment; N (%) Nephropathy Microalbuminuria Associated conditions; N (%) Hypothyroidism Autoimmune hepatitis
P value
Negative
Positive
10.7 3.8 3.04 1.98
9.3 3.6 2.8 1.3
0.2 0.7
15 (57.7) 11 (42.3)
7 (36.8) 12 (63.2)
0.2
25 (96.2) 1 (3.8)
19 (100) 0
0.9
12 (46.2) 11 (42.3) 12 (46.2)
12 (63.2) 9 (47.4) 8 (42.1)
0.5
0 1 (3.8)
1 (5.3) 0
0.3
0 1 (3.8)
1 (5.3) 0
0.3
0.8
4. Discussion The current study shows that the overall frequency of MEFV gene mutations was 42% in diabetics and 34.1% in healthy subjects with no significant difference. Only the diabetic group showed compound heterozygous gene mutations (11% vs 0%). The commonest MEFV gene mutations in the diabetic children are: E148Q in 7 children, A744S in 3 children, V726A in 2 children, M680I (G/C) in
Table 4 Comparison between diabetic children with heterozygous and compound heterozygous gene mutations.
Age; mean SD DM duration; mean SD Sex; N (%) Male Female Consanguinity; N (%) Nephropathy; N (%) Hypothyroidism; N (%) History; N (%) Abdominal pain Arthralgia
Heterozygous n ¼ 14
Compound heterozygous n ¼ 5
P value
8.6 3.7 2.9 1.4
11.4 2.4 2.8 1.3
0.08 0.9
6 8 6 0 1
1 4 2 1 0
0.6
(42.9) (57.1) (42.9) (7.1)
10 (71.4) 6 (42.9)
(20) (80) (40) (20)
2 (40) 3 (60)
0.9 NA NA 0.2 0.9
0 1
1 0
1 0
1 1
1 child and P369S in1 child, while 5 children (11.1%) were compound heterozygous (M694V/M694I in 2 children, E148Q/K695R mutations in 1 child, E148Q/M694I in 1 child and E148Q/V726A in 1 child). A possible association of FMF with type 1 diabetes can be explained by that immune dysregulation in FMF may be involved in the autoimmune mechanism that leads to type 1 diabetes [Bas¸ et al., 2009]. This hereditary (monogenic) autoinflammatory disease is often associated with mutations in one of several proteins involved in innate immune pathways [Ozkurede and Franchi, 2012]. Gulcan et al. [Gulcan et al., 2009] reported a 21-year-old woman who had FMF with Hashimoto’s thyroiditis [HT]. They argued that FMF and HT have similar pathophysiological mechanisms, and cytokine expression in FMF may provoke an autoimmune response. From the pathogenetic point of view, both autoinflammatory diseases (AIDs) and autoimmune diseases (AD) are characterized by a chronic activation of immune system, which eventually leads to tissue inflammation in genetically predisposed individuals. In AIDs the innate immune system directly causes tissue inflammation, whereas in ADs the innate immune system activates the adaptive immune system which, in turn, is responsible for the inflammatory process. Mutations in inflammasome-related proteins, particularly in NOD-like receptor (NLR) genes, have been strongly associated to the occurrence of AIDs. A role for this multiprotein-complex in some ADs can be postulated, since a wide spectrum of endogenous danger signals can activate NLRs and inflammasome products, including IL-1b, can activate adaptive immunity. AIDs and ADs might be considered as a single group of diseases with a large immune pathological and clinical spectrum which includes at one end pure ADs and at the other end pure AIDs [Doria et al., 2012]. Up to our knowledge, this the first report studied the FMF gene mutations in type 1 diabetic children. The carrier rate in the current study was 42% in diabetic group Vs 34% in the control group with no statistical significance (P ¼ 0.09). Both diabetic and control subjects had high positive consanguinity (44% Vs 42%). It seems that the high prevalence of MEFV gene mutations is our study group is related to high rate of positive consanguinity in our country. Moreover, the small difference in carrier rate in both groups may well be explained by the difference in consanguinity rate, a coincidence, bias or a combination of these three. The carrier rate in the current study is higher than that was reported by Al-Alami et al. [Al-Alami et al., 2003] in normal Egyptian population; which was 16%. E148Q gene mutation is the most prevalent in the current study. Our results compared to those of Al Garf et al. [El-Garf et al., 2010]; who studied the same 12 mutation analysis in a group of Egyptian children with FMF; showed that the most frequent gene mutation was V726A in 41.2%, followed by M694V, M680I, E148Q and M694I in 32.4, 29.4, 25 and 20.6%, respectively. This difference in frequency of gene mutation may be due to reduced penetrance of this mutation (E148Q) or it may be a polymorphism and explains the fact that a considerable proportion of the genetically affected individuals remain asymptomatic. Previous reports indicated that, individuals who were homozygous or compound heterozygous for E148Q, may lack clinical features [Yilmaz et al., 2001].
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G.M. Anwar et al. / European Journal of Medical Genetics 58 (2015) 31e34
Our results come in agreement with those of Belmahi et al. [Belmahi et al., 2006] who found that the most frequent mutations among Arabs of North Africa were M694V and M694I. Comparison between diabetic patients with negative and positive gene mutation and those with heterozygous and compound heterozygous gene mutations showed no significant difference regarding abdominal pain, fever, arthralgia and nephropathy. This is mainly attributed to the high frequency of mutations of low penetrance (E148Q, V726A) and partly to the role of unknown, genetic and/or environmental, modifiers. Recent population based studies have shown that the frequency of FMF mutant alleles by far exceed those deduced from the prevalence of the disease and assert that the majority of individuals, who comply with the genetic definition of FMF, remain unaffected [Gershoni-Baruch et al., 2001; Kogan et al., 2001; Stoffman et al., 2000]. M694V homozygosity has been reported to have more severe disease and a higher risk of amyloidosis than those who are not carriers of this mutation [Dewalle et al., 1998; Livneh et al., 1999]. E148Q mutation is generally accepted to be associated with mild disease [Mimouni et al., 2000], other authors consider it to be a polymorphism rather than a disease causing mutation, and the presence of homozygosity for E148Q may not be sufficient for developing clinical disease [Tchernitchko et al., 2003]. Amyloidosis may develop in FMF patients who do not have attacks of serositis [Gershoni-Baruch et al., 2002]. 5. Conclusion Our results affirm the high carrier rate of MEFV gene mutations among the screened Egyptian children with type 1 diabetes and in controls as well. Only the diabetics group showed compound heterozygous MEFV gene mutations. Egyptian population have high carrier rate of MEFV gene mutations due to high consanguinity rate. Funding resources National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt. Conflict of interest No conflict of interest to declare. Acknowledgements This study has been funded partially by National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt in the form of kits for testing MEFV gene. References Aksentijevich I, Torosyan Y, Samuels J, et al. Mutation and haplotype studies of familial Mediterranean fever reveal new ancestral relationships and evidence for a high carrier frequency in the Ashkenazi Jewish population. Am J Hum Genet 1999;64:949e62. Aksu K, Keser G. Coexistence of vasculitides with familial Mediterranean fever. Rheumatol Int 2011;31:1263e74. Al-Alami JR, Tayeh MK, Najib DA, Abu-Rubaiha ZA, Majeed HA, El-Khateeb MS, ElShanti HI. Familial Mediterranean fever mutation frequencies and carrier rates among a mixed Arabic population. Saudi Med J 2003;24:1055e9. Atabek ME, Pirgon O, Sert A, Arslan U. Familial Mediterranean fever associated with type 1 diabetes. Endocrinol 2006;16:133e5. Bas¸ F, Kabatas¸-Eryılmaz S, Günöz H, Darendeliler F, Küçükemre B, Bundak R, Saka N. Type 1 diabetes mellitus associated with autoimmune thyroid disease,
celiac disease and familial Mediterranean fever: case report. Turk J Ped 2009;51:183e6. Belmahi L, Se W ani A, Feingold J, Delpech M, Grateau G, Dode C. Prevalence and distribution of MEFV mutations among Arabs from the Maghreb patients su V ering from familial Mediterranean fever. C R Biol 2006;329:71e4. Ben-Chetrit E. Familial Mediterranean fever and renal AA amyloidosis. Phenotypegenotype correlation, treatment and prognosis. J Nephrol 2003;16:431e4. Bernot A, da Saliva C, Petit J-L, et al. Non-founder mutations in the MEFV gene establish this gene as the cause of familial Mediterranean fever. Hum Mol Genet 1998;7:1317e25. Cazeneuve C, Sarkisian T, Pecheux C, et al. MEFV-gene analysis in Armenian patients with familial Mediterranean fever: diagnostic value and unfavourable renal prognosis of the M694V homozygous genotype-genetic and therapeutic implications. Am J Hum Genet 1999;65:88e97. Dewalle M, Domingo C, Rozenbaum M, Ben-Chetrit E, Cattan D, Bernot A, et al. Phenotype-genotype correlation in Jewish patients suffering from familial Mediterranean fever (FMF). Eur J Hum Genet 1998;6:95e7. Dodé C, Pécheux C, Cazeneuve C, et al. Mutations in the MEFV gene in a large series of patients with a clinical diagnosis of familial Mediterranean fever. Am J Med Genet 2000;92:241e6. Doria A, Zen M, Bettio S, Gatto M, Bassi N, Nalotto L, Ghirardello A, Iaccarino L, Punzi L. Autoinflammation and autoimmunity: bridging the divide. Autoimmun Rev 2012;12:22e30. http://dx.doi.org/10.1016/j.autrev.2012.07.018. El-Garf A, Salah S, Iskander I, Salah H, Amin SN. MEFV mutations in Egyptian patients suffering from familial Mediterranean fever: analysis of 12 gene mutations. Rheumatol Int 2010;30:1293e8. Ergul AB, Dursun I, Torun YA. Hashimoto’s thyroiditis in a child with familial mediterranean fever: a case report. Iran J Pediatr 2013;23:489e90. Erten S, Taskaldiran I, Yakut ZI. Are systemic lupus erythematosus patients carrying MEFV gene less prone to renal involvement? Report of three cases and review of the literature. Ren Fail 2013;35:1013e6. FMF International Consortium. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever. Cell 1997;90:797e807. Gershoni-Baruch R, Shinawi M, Leah K, Badarnah K, Brik R. Familial Mediterranean fever: prevalence, penetrance and genetic drift. Eur J Hum Genet 2001;9:634e7. Gershoni-Baruch R, Brik R, Shinavi M, Livneh A. The different contribution of MEFV mutant alleles to the clinical profile of familial Mediterranean fever. Eur J Hum Genet 2002;10:145e9. Gulcan E, Gulcan A, Koplay M, Alcelik A, Korkmaz U. Co-existence of Hashimoto’s thyroiditis with familial Mediterranean fever: is there a pathophysiological association between the two diseases? Clin Exp Immunol 2009;156:373e6. http://dx.doi.org/10.1111/j.1365-2249.2009.03891.x. Kogan A, Shinar Y, Lidar M, et al. Common MEFV mutations among Jewish ethnic groups in Israel: high frequency of carrier and phenotype III states and absence of a perceptible biological advantage for the carrier state. Am J Med Genet 2001;102:272e6. Livneh A, Langevitz P, Shinar Y, Zaks N, Kastner DL, Pras M, et al. MEFV mutation analysis in patients suffering from amyloidosis of familial Mediterranean fever. Amyloid 1999;6:1e6. Mimouni A, Magal N, Stoffman N, Shohat T, Minasian A, Krasnov M, et al. Familial Mediterranean fever: effects of genotype and ethnicity on in X ammatory attacks and amyloidosis. Pediatrics 2000;105:E70. Ozkurede VU, Franchi L. Immunology in clinic review series; focus on autoinflammatory diseases: role of inflammasomes in autoinflammatory syndromes. Clin Exp Immunol 2012;167:382e90. http://dx.doi.org/10.1111/j.13652249.2011.04535.x. Pras E, Aksentijevich I, Gruberg L, Balow Jr JE, Prosen L, Dean M. Mapping of a gene causing familial Mediterranean fever to the short arm of chromosome 16. N Engl J Med 1992;326:1509e13. Ritis K, Giaglis S, Spathari N, Micheli A, Zonios D, Tzoanopoulos D, et al. 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 2004;63:438e43. Shohat M, Halpern GJ. Familial Mediterranean fever-a review. Genet Med 2011;13: 487e98. Stoffman N, Magal N, Shohat T, Lotan R, Koman S, Oron A, et al. Higher than expected carrier rates for familial Mediterranean fever in various Jewish ethnic groups. Eur J Hum Genet 2000;8:307e10. Tchernitchko D, Legendre M, Cazeneuve C, Delahaye A, Neil F, Amselen S. The E148Q MEFV allele is not implicated in the development of familial Mediterranean fever. Hum Mutat 2003;22:239e40. Yildiz G, Kayatas M, Uygun Y, Timucin M, Candan F. Coexistence of systemic lupus erythematosus and familial mediterranean fever. Intern Med 2010;49:767e9. Yilmaz E, Ozen S, Balcı B, Duzova A, Topaloglu R, Besbas N, Saatci U, Bakkaloglu A, Ozguc M. Mutation frequency of Familial Mediterranean Fever and evidence for a high carrier rate in the Turkish population. Eur J Hum Genet 2001;9:553e5.
Contents lists available at ScienceDirect
European Journal of Medical Genetics journal homepage: http://www.elsevier.com/locate/ejmg
Clinical research
A study of familial Mediterranean Fever (MEFV) gene mutations in Egyptian children with type 1 diabetes mellitus Ghada Mohammad Anwar a, Hanan M. Fouad b, *, Amal Abd El-Hamid b, Faten Mahmoud c, Noha Musa a, Hala Lotfi a, Nermine Salah a a b c
Pediatric Department, Cairo University, Cairo, Egypt Pediatric Department, National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt Chemical Department, National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history: Received 10 July 2014 Accepted 19 October 2014 Available online 4 November 2014
Background/Aims: An association of type 1 DM and familial Mediterranean fever (FMF) has been newly reported in the medical literature. The aim of the present work was to investigate frequency of MEFV gene mutations in Egyptian children with type 1 diabetes mellitus. Methods: Forty five children with type 1 DM were screened for Mediterranean Fever (MEFV) gene mutation. Forty one healthy control subjects were included. Identification of FMF gene mutation was done based on polymerase chain reaction (PCR) and reverse hybridization. The assay covers 12 mutations in the FMF gene: E148Q e P369S e F479L e M680I (G/C) e M680I (G/A) e I692del e M694V e M694I e K695ReV726A e A744S and R761H. Results: Among the screened diabetics, the overall frequency of MEFV gene mutations was 42.2% and among the control group it was 34.1% with no significant difference. Fourteen out of 45 diabetic children (31.1%) were heterozygous (E148Q in 7 children, A744S in 3 children, V726A in 2 children, M680I (G/C) in 1 child and P369S in1 child), while 5 children (11.1%) were compound heterozygous (M694V/M694I in 2 children, E148Q/K695R mutations in 1 child, E148Q/M694I in 1 child and E148Q/V726A in 1 child). The control group showed heterozygous mutation in 34.1% of cases (E148Q mutation in 14.6%, V726A in 12.2%, M680I (G/C) in 4.9% and M694V in 2.4%). Conclusion: No significant difference in mutation frequency between diabetic and non-diabetic children. We have high carrier rate of MEFV gene mutations among Egyptian population probably due to high consanguinity. Ó 2014 Elsevier Masson SAS. All rights reserved.
Keywords: Type 1 diabetes Familial Mediterranean fever Children MEFV gene mutation
1. Introduction Familial Mediterranean fever (FMF) is an autoinflammatory diseases that is often associated with other autoimmune diseases such as ankylosing spondylitis, rheumatoid arthritis, polyarteritis nodosa (PAN), Behcet, multiple sclerosis (MS), and Systemic Lupus (SLE), Henoch-Schönlein purpura, and Hashimoto’s thyroiditis [Yildiz et al., 2010; Aksu and Keser, 2011; Erten et al., 2013; Ergul et al., 2013]. An association of type 1 DM and FMF has been newly reported in the medical literature [Atabek et al., 2006; Bas¸ et al., 2009]. FMF is an autosomal recessive disorder and it comprises two phenotypes: FMF type 1 is characterized by recurrent short
* Corresponding author. E-mail address: [email protected] (H.M. Fouad). http://dx.doi.org/10.1016/j.ejmg.2014.10.005 1769-7212/Ó 2014 Elsevier Masson SAS. All rights reserved.
episodes of inflammation and serositis including fever, peritonitis, synovitis, pleuritis, and rarely, pericarditis and meningitis. FMF type 2 is characterized by amyloidosis as the first clinical manifestation of FMF in an otherwise asymptomatic individual [Shohat and Halpern, 2011]. In 1997 the gene responsible for FMF, MEFV, was identified [Ritis et al., 2004; FMF International Consortium, 1997]. FMF is due to recessive mutations in the MEFV (for Mediterranean Fever) gene, located on the short arm of the chromosome 16 (16p13.3), consisting of 10 exons and 781 codons [Pras et al., 1992]. The type and combination of the mutations define a severely or weakly expressed phenotype [Ben-Chetrit, 2003; Dodé et al., 2000; Gershoni-Baruch et al., 2002], but, on the other hand, subjects carrying two mutations may not express the disease [Kogan et al., 2001]. Mutations in the pyrin/marenostrin (MEFV) gene have been identified in the majority of FMF patients [Aksentijevich et al., 1999;
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G.M. Anwar et al. / European Journal of Medical Genetics 58 (2015) 31e34
Bernot et al., 1998]. 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 vast majority of FMF chromosomes identified [Aksentijevich et al., 1999; Bernot et al., 1998; Gershoni-Baruch et al., 2001; Kogan et al., 2001]. It has been established, that the phenotypic variability of the disease is, at least, partly due to allelic heterogeneity. Mutation M694V is associated with a severe phenotype and amyloidosis, and mutation V726A with a milder form of the disease [Cazeneuve et al., 1999; Dewalle et al., 1998]. The observation that V726A e E148Q allele is associated with a severe disease and strongly predisposes to renal amyloidosis, highlights the need to prescribe colchicine, as early as possible, to patients who carry the complex allele. Since amyloidosis, which is preferentially associated with either the M694V or the V726A e E148Q allele, may develop in FMF patients who do not have attacks of serositis, the need to screen asymptomatic family members, for mutations in the MEFV gene, is underlined [Gershoni-Baruch et al., 2002]. Up to our knowledge, there are no previous researches investigated the frequency of MEFV gene mutation in Egyptian type 1 diabetic patients. The aim of the present work was to investigate frequency of 12 MEFV gene mutations in Egyptian children with type 1 diabetes mellitus. 2. Subjects and methods Forty five children with type 1 DM, attending the Diabetes Endocrine and Metabolism Pediatric Unit at Cairo University Pediatric Hospital were selected randomly and screened for Mediterranean Fever (MEFV) gene mutation. Inclusion criteria were Diabetic children of any age and both sexes. Approval by the ethical committee was obtained. Enrollment was done after obtaining verbal consent from parents. Enrolled children were subjected to medical history (age, sex, consanguinity of parents, duration of diabetes, fever, abdominal pain, arthralgia, pleurisy, myalgia, rash), medical examination (for different systems; joints, chest, cardiovascular system, central nervous system, abdomin), routine laboratory investigations including HbA1c, thyroid profile, and urinary albumin/creatinine ratio were done in all patients and blood samples were obtained for assay of FMF gene mutation. Exclusion criteria were any patient who was already diagnosed as FMF or other rheumatic disorder. Forty one healthy; age and sex matched; children were included as a control group.
frequency and percentage. Chi-square test, unpaired t-student test were used to compare children with and those without MEFV gene mutation. Level of significance was P 0.05 for all statistical analyses. 3. Results Among the 45 diabetic children included in the study, 49% were male and 51% were females. The mean age of the children was 10.1 3.7 years (2.5e17), and the mean duration of diabetes was 3 1.7 years (1e8), mean HbA1c was 8.23 1.48, and the mean insulin dose was 1.19 0.28 U/Kg/day. Positive consanguinity of parents was detected in 44.4%. Overt nephropathy was detected in one patient, microalbuminuria in one patient, and hypothyroidism in one patient. Descriptive data of the diabetic group was summarized in Table 1. Among the control group, 44% were male and 56% were females and their ages ranged from 5 to 16 years. Positive consanguinity of parents was detected in 42%. Among the screened diabetic children, 19 (42.2%) were positive for MEFV gene mutations, while 26 (57.8%) were negative. Fourteen out of 45 children (31.1%) were heterozygous (E148Q in 7 children, A744S in 3 children, V726A in 2 children, M680I (G/C) in 1 child and P369S in1 child), while 5 children (11.1%) were compound heterozygous (M694V/M694I in 2 children, E148Q/K695R in 1 child, E148Q/M694I in 1 child and E148Q/V726A in 1 child) (Table 2). Among the screened control subjects, 14 (34%) were positive for MEFV gene mutations, while 27 (66%) were negative, with no significant difference between diabetic and control subjects (P ¼ 0.09). In the control group, all subjects were heterozygous (E148Q in 6 children, V726A in 5 children, M680I in 2 children, and M694V in one child). A comparison between diabetic and control subjects regarding MEFV gene mutations is shown in Table 2. Comparison of diabetic children with positive and negative MEFV gene mutation showed no significant difference in both groups regarding recurrent abdominal pain, arthralgia, nephropathy, microalbuminuria, hypothyroidism and hepatomegaly due to autoimmune hepatitis (Table 3). Comparison between patients with heterozygous mutation and compound heterozygous gene mutation was shown in Table 4. Overt nephropathy was found in one patient in compound heterozygous group (M694V/M694I). One patient with heterozygous MEFV gene mutation has family history of type 1 diabetes. Compound heterozygous mutation was detected in 2 patients with positive consanguinity as well as 3 patients with negative consanguinity, mutation types are shown in Table 5.
2.1. FMF gene mutation analysis A 5 ml EDTA blood sample was obtained by venipuncture for FMF assay. Identification of FMF gene mutation was done based on polymerase chain reaction (PCR) and reverse hybridization. Procedure includes three steps: DNA isolation, PCR amplification using biotinylated primers and hybridization of the amplification products to a test strip containing allele specific oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences are detected using streptavidin alkaline phosphatase and color substrate. The assay covers 12 mutations in the FMF gene: E148Q e P369S e F479L e M680I (G/C) e M680I (G/A) e I692del e M694V e M694I e K695ReV726A e A744S and R761H. 2.2. Statistical analysis Data were analyzed by Statistical Package for Social Science (SPSS) program version 17.0. Descriptive statistics such as mean (standard deviation) for normally distributed data were calculated for quantitative parameters. Qualitative data were summarized as
Table 1 Descriptive data of the diabetic group. Data Sex Male Female Residence Urban Rural Positive consanguinity History Abdominal pain Arthralgia Hepatomegaly Renal assessment Overt Nephropathy Microalbuminuria Associated conditions Hypothyroidism Autoimmune hepatitis
No (%) 22 (48.9) 23 (51.1) 44 (97.8) 1 (2.2) 20 (44.4) 24 (39.3) 20 (32.8) 1 (2.2) 1 (2.2) 1 (2.2) 1 (2.2) 1 (2.2)
G.M. Anwar et al. / European Journal of Medical Genetics 58 (2015) 31e34 Table 2 Comparison of MEFV gene mutation between diabetic and control subjects.
33
Table 5 Mutation types in consanguineous and non-consanguineous parents.
MEFV gene mutation
Diabetic children N ¼ 41
Controls N ¼ 41
P value
Consanguinity E148Q - K695R E148Q-V726A E148V-M694I M694V - M694I
Negative Heterozygous; E148Q A744S V726A M680I (G/C) P369S M694V Compound heterozygous; M694V/M694I E148Q/K695R E148Q/M694I E148Q/V726A
26 (57.8) 14 (31.1) 7 (15.6) 3 (6.7) 2 (4.4) 1 (2.2) 1 (2.2) 0 5 (11.1) 2 (4.4) 1 (2.2) 1 (2.2) 1 (2.2)
27 (65.9) 14 (34.1) 6 (14.6) 0 5 (12.2) 2 (4.9) 0 1 (2.4) 0
0.09
Negative Positive
Table 3 Comparison between diabetic children with positive and negative MEFV gene mutation. MEFV gene mutation
Age; Mean SD Duration of DM; Mean SD Sex; N (%) Male Female Residence; N (%) Urban Rural History; N (%) Abdominal pain Arthralgia Positive consanguinity; N (%) Renal assessment; N (%) Nephropathy Microalbuminuria Associated conditions; N (%) Hypothyroidism Autoimmune hepatitis
P value
Negative
Positive
10.7 3.8 3.04 1.98
9.3 3.6 2.8 1.3
0.2 0.7
15 (57.7) 11 (42.3)
7 (36.8) 12 (63.2)
0.2
25 (96.2) 1 (3.8)
19 (100) 0
0.9
12 (46.2) 11 (42.3) 12 (46.2)
12 (63.2) 9 (47.4) 8 (42.1)
0.5
0 1 (3.8)
1 (5.3) 0
0.3
0 1 (3.8)
1 (5.3) 0
0.3
0.8
4. Discussion The current study shows that the overall frequency of MEFV gene mutations was 42% in diabetics and 34.1% in healthy subjects with no significant difference. Only the diabetic group showed compound heterozygous gene mutations (11% vs 0%). The commonest MEFV gene mutations in the diabetic children are: E148Q in 7 children, A744S in 3 children, V726A in 2 children, M680I (G/C) in
Table 4 Comparison between diabetic children with heterozygous and compound heterozygous gene mutations.
Age; mean SD DM duration; mean SD Sex; N (%) Male Female Consanguinity; N (%) Nephropathy; N (%) Hypothyroidism; N (%) History; N (%) Abdominal pain Arthralgia
Heterozygous n ¼ 14
Compound heterozygous n ¼ 5
P value
8.6 3.7 2.9 1.4
11.4 2.4 2.8 1.3
0.08 0.9
6 8 6 0 1
1 4 2 1 0
0.6
(42.9) (57.1) (42.9) (7.1)
10 (71.4) 6 (42.9)
(20) (80) (40) (20)
2 (40) 3 (60)
0.9 NA NA 0.2 0.9
0 1
1 0
1 0
1 1
1 child and P369S in1 child, while 5 children (11.1%) were compound heterozygous (M694V/M694I in 2 children, E148Q/K695R mutations in 1 child, E148Q/M694I in 1 child and E148Q/V726A in 1 child). A possible association of FMF with type 1 diabetes can be explained by that immune dysregulation in FMF may be involved in the autoimmune mechanism that leads to type 1 diabetes [Bas¸ et al., 2009]. This hereditary (monogenic) autoinflammatory disease is often associated with mutations in one of several proteins involved in innate immune pathways [Ozkurede and Franchi, 2012]. Gulcan et al. [Gulcan et al., 2009] reported a 21-year-old woman who had FMF with Hashimoto’s thyroiditis [HT]. They argued that FMF and HT have similar pathophysiological mechanisms, and cytokine expression in FMF may provoke an autoimmune response. From the pathogenetic point of view, both autoinflammatory diseases (AIDs) and autoimmune diseases (AD) are characterized by a chronic activation of immune system, which eventually leads to tissue inflammation in genetically predisposed individuals. In AIDs the innate immune system directly causes tissue inflammation, whereas in ADs the innate immune system activates the adaptive immune system which, in turn, is responsible for the inflammatory process. Mutations in inflammasome-related proteins, particularly in NOD-like receptor (NLR) genes, have been strongly associated to the occurrence of AIDs. A role for this multiprotein-complex in some ADs can be postulated, since a wide spectrum of endogenous danger signals can activate NLRs and inflammasome products, including IL-1b, can activate adaptive immunity. AIDs and ADs might be considered as a single group of diseases with a large immune pathological and clinical spectrum which includes at one end pure ADs and at the other end pure AIDs [Doria et al., 2012]. Up to our knowledge, this the first report studied the FMF gene mutations in type 1 diabetic children. The carrier rate in the current study was 42% in diabetic group Vs 34% in the control group with no statistical significance (P ¼ 0.09). Both diabetic and control subjects had high positive consanguinity (44% Vs 42%). It seems that the high prevalence of MEFV gene mutations is our study group is related to high rate of positive consanguinity in our country. Moreover, the small difference in carrier rate in both groups may well be explained by the difference in consanguinity rate, a coincidence, bias or a combination of these three. The carrier rate in the current study is higher than that was reported by Al-Alami et al. [Al-Alami et al., 2003] in normal Egyptian population; which was 16%. E148Q gene mutation is the most prevalent in the current study. Our results compared to those of Al Garf et al. [El-Garf et al., 2010]; who studied the same 12 mutation analysis in a group of Egyptian children with FMF; showed that the most frequent gene mutation was V726A in 41.2%, followed by M694V, M680I, E148Q and M694I in 32.4, 29.4, 25 and 20.6%, respectively. This difference in frequency of gene mutation may be due to reduced penetrance of this mutation (E148Q) or it may be a polymorphism and explains the fact that a considerable proportion of the genetically affected individuals remain asymptomatic. Previous reports indicated that, individuals who were homozygous or compound heterozygous for E148Q, may lack clinical features [Yilmaz et al., 2001].
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G.M. Anwar et al. / European Journal of Medical Genetics 58 (2015) 31e34
Our results come in agreement with those of Belmahi et al. [Belmahi et al., 2006] who found that the most frequent mutations among Arabs of North Africa were M694V and M694I. Comparison between diabetic patients with negative and positive gene mutation and those with heterozygous and compound heterozygous gene mutations showed no significant difference regarding abdominal pain, fever, arthralgia and nephropathy. This is mainly attributed to the high frequency of mutations of low penetrance (E148Q, V726A) and partly to the role of unknown, genetic and/or environmental, modifiers. Recent population based studies have shown that the frequency of FMF mutant alleles by far exceed those deduced from the prevalence of the disease and assert that the majority of individuals, who comply with the genetic definition of FMF, remain unaffected [Gershoni-Baruch et al., 2001; Kogan et al., 2001; Stoffman et al., 2000]. M694V homozygosity has been reported to have more severe disease and a higher risk of amyloidosis than those who are not carriers of this mutation [Dewalle et al., 1998; Livneh et al., 1999]. E148Q mutation is generally accepted to be associated with mild disease [Mimouni et al., 2000], other authors consider it to be a polymorphism rather than a disease causing mutation, and the presence of homozygosity for E148Q may not be sufficient for developing clinical disease [Tchernitchko et al., 2003]. Amyloidosis may develop in FMF patients who do not have attacks of serositis [Gershoni-Baruch et al., 2002]. 5. Conclusion Our results affirm the high carrier rate of MEFV gene mutations among the screened Egyptian children with type 1 diabetes and in controls as well. Only the diabetics group showed compound heterozygous MEFV gene mutations. Egyptian population have high carrier rate of MEFV gene mutations due to high consanguinity rate. Funding resources National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt. Conflict of interest No conflict of interest to declare. Acknowledgements This study has been funded partially by National Hepatology and Tropical Medicine Research Institute, Cairo, Egypt in the form of kits for testing MEFV gene. References Aksentijevich I, Torosyan Y, Samuels J, et al. Mutation and haplotype studies of familial Mediterranean fever reveal new ancestral relationships and evidence for a high carrier frequency in the Ashkenazi Jewish population. Am J Hum Genet 1999;64:949e62. Aksu K, Keser G. Coexistence of vasculitides with familial Mediterranean fever. Rheumatol Int 2011;31:1263e74. Al-Alami JR, Tayeh MK, Najib DA, Abu-Rubaiha ZA, Majeed HA, El-Khateeb MS, ElShanti HI. Familial Mediterranean fever mutation frequencies and carrier rates among a mixed Arabic population. Saudi Med J 2003;24:1055e9. Atabek ME, Pirgon O, Sert A, Arslan U. Familial Mediterranean fever associated with type 1 diabetes. Endocrinol 2006;16:133e5. Bas¸ F, Kabatas¸-Eryılmaz S, Günöz H, Darendeliler F, Küçükemre B, Bundak R, Saka N. Type 1 diabetes mellitus associated with autoimmune thyroid disease,
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