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DQ polymorphism with myasthenia gravis in Tunisian patients
Clinical Neurology and Neurosurgery 115 (2013) 32–36
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Association of HLA-DR/DQ polymorphism with myasthenia gravis in Tunisian patients Najiba Fekih-Mrissa a,∗ , Sarra Klai a , Jamel Zaouali b , Nasreddine Gritli a , Ridha Mrissa b a b
Laboratory of Molecular Biology, Department of Hematology, Military Hospital, Tunis, Tunisia Department of Neurology, Military Hospital, Tunis, Tunisia
a r t i c l e
i n f o
Article history: Received 25 May 2011 Received in revised form 16 January 2012 Accepted 1 April 2012 Available online 18 April 2012 Keywords: HLA class II polymorphism Myasthenia gravis
a b s t r a c t Background and objective: Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction. MG has been shown to be associated with many human leukocyte antigens (HLA) in different populations. The aim of this study was to investigate the probable association between HLA-DR/DQ alleles and MG in Tunisian patients. Patients and methods: HLA DR/DQ genotyping was performed using polymerase chain reaction sequencespecific primers (PCR-SSP) with 48 MG patients and 100 healthy individuals serving as the control group. Results: Myasthenia gravis in Tunisian patients was found to be associated with the following alleles (pc denotes Bonferroni corrected probability values): HLA-DRB1*03 (pc < 10−3 ), DRB1*04 (pc = 0.005), DQB1*02 (pc = 0.002) and, DQB1*03 (pc = 0.007). Conclusion: Our data demonstrated a new HLA-MG predisposition with DRB1*04. The DRB1*03, DRB1*04, DQB1*02, and DQB1*03 alleles also could be predisposing genetic factors for MG in the Tunisian population. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Myasthenia gravis (MG) is a rare and heterogeneous autoimmune disease characterized by progressive weakness and fatigue of the voluntary musculature. It is caused by immune-mediated loss of functioning acetylcholine receptors (AChR) from the post-synaptic membrane of the neuromuscular junction [1,2]. The association with human leukocyte antigens (HLA) has been known since the early 1970s [3,4]. These antigens strongly influence susceptibility to the development of MG [5,6]. In Caucasian populations, the most commonly cited alleles in the HLA complex that have been associated with MG are DRB1*03, DQB1*02, and DQB1*03 [7,8]. Studies of different populations have revealed that the frequencies of different HLA antigens vary depending on sex, age at disease onset, ethnic, and geographic origins of these populations [9].
Abbreviations: AChR, acetylcholine receptors; EMG, electromyography; HLA, human leukocyte antigens; LD, linkage disequilibrium; MG, myasthenia gravis; PCR, polymerase chain reaction; SSP, sequence-specific primer. ∗ Corresponding author at: Laboratory of Molecular Biology, Department of Hematology, Military Hospital. 1008 Mont Fleury, Tunis, Tunisia. Tel.: +216 22510488; fax: +216 70762084. E-mail address: [email protected] (N. Fekih-Mrissa). 0303-8467/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clineuro.2012.04.001
In this study, we have genotyped HLA class II alleles in Tunisian MG patients and examined HLA class II association with MG subjects to understand HLA genotype effects on disease pathogenesis.
2. Methods 2.1. Subjects Forty-eight (22 women and 26 men) unrelated Tunisian MG patients were studied (male to female ratio = 1.18). The patients were referred from all regions of the country and were selected to have the generalized type of myasthenia; all other types were excluded. Myasthenia gravis was diagnosed according to conventional clinical criteria, electromyography, and/or a positive acetylcholine receptor antibodies test. The neostigmine test was positive for all patients. In AChR seronegative patients, the diagnosis was confirmed by electromyography and the positive neostigmine test. The main characteristics are summarized in Table 1. Serum anti-AChR antibody titer (nmol/L) was determined at the time of diagnosis using a conventional radioimmunoprecipitation assay. The controls consisted of 100 healthy unrelated individuals (48 women and 52 men) with diverse Tunisian origin similar to the patients (male to female ratio = 1.08). All participants gave informed consent before participation in the study.
N. Fekih-Mrissa et al. / Clinical Neurology and Neurosurgery 115 (2013) 32–36 Table 1 Characteristics of myasthenia gravis patients. Characteristics Sex Female Male Age at onset <40 Y >40 Y Thymoma EMGa (+) (−) Anti-AChr Abb (+) (−)
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3.2. HLA-DRB/DQB allele
Number of patients (n = 48)
Percentage
22 26
45.8 54.2
30 18 18
62.5 37.5 37.5
42 6
87.5 12.5
28 20
58.3 41.7
a EMG, electromyography on the extremity muscle; (+), myasthenic pattern positive, (−), myasthenic pattern negative.. b Anti-AChR Ab, anti-acetylcholine receptors antibody; (+), over 0.2 nm, (−), under 0.2 nm.
2.2. HLA-typing by DNA amplification Genomic DNA was extracted from peripheral blood samples of patients and healthy individuals using the QIAamp® DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany). Low-resolution HLA typing was performed by PCR-SSP techniques according to Micro SSP DNA Typing Trays DRB/DQB (One Lambda Inc. Canoga Park, CA, USA). Amplified DNA fragments were detected by agarose gel electrophoresis (2.5% agarose gel), stained with ethidium bromide, and UV transillumination. One Lambda DNA/LMT software version 3.98 was used to detect specific DRB1 and DQB1 alleles.
2.3. Statistical analysis Allele frequencies were estimated by using the direct counting method. Haplotype frequencies were estimated using the EM algorithm and deviations from Hardy–Weinberg equilibrium were both performed using the Arlequin v.3.1 software (http://cmpg.unibe.ch/software/arlequin3). All analyses related to the case–control study were performed using the Statistical Package for the Social Sciences v.16 (SPSS Inc., Chicago, IL). Differences between cases and controls were evaluated by using the chisquare test or Fisher’s test for qualitative variables and t-test for quantitative variables. The odds ratios (OR) and 95% confidence intervals (CI) were also calculated. The Bonferroni method was used to adjust for type I errors due to multiple comparisons. Corrected probability values (pc ) were calculated for the number of comparisons made, i.e., the number of alleles tested. Probability values pc < 0.05 were considered statistically significant.
Table 2 (section a) shows the frequencies of DRB1, 3, 4 and 5 alleles in patients and controls. The prevalence of DRB1*03 and DRB1*04 was more elevated in MG patients than in controls (30.2% vs. 11.5%) and (28.12% vs. 14.5%). Both types DRB1*03 and DRB1*04 were positively and significantly associated with patients (pc < 10−3 , pc = 0.005 respectively). Both DRB3*01 and DRB4*01 were found to be in linkage disequilibrium (LD) with DRB1*03 and DRB1*04 respectively. The prevalence of types DRB3*01 and DRB4*01 in MG patients therefore can be explained by high frequencies of types DRB1*03 and DRB1*04, respectively. Table 2 (section b) shows the frequencies of the HLA DQB1 allele in patients and controls. Both DQB1*02 (41.6% vs. 24.5%, pc = 0.002) and DQB1*0302 (26.0% vs. 13.5%, pc = 0.007) were positively associated with MG. Table 3 shows the frequencies of HLA DR/DQ allele haplotypes in patients and control subjects. Only haplotypes exhibiting significant LD parameters between alleles were considered for study. The calculation of the LD parameter of two locus haplotypes, in both cases and controls, revealed that the most significant values of the LD parameters were for the pairs DRB1*03/DQB1*02 and DRB1*04/DQB1*0302. Compared with controls, patients showed a higher frequency of haplotypes DRB1*03/DQB1*02 (26.0% of patients vs. 9% of controls, pc < 10−3 ) and haplotype DRB1*04/DQB1*0302 (19.7% of patients vs. 8% of controls, pc = 0.004). Table 4 shows the frequency of significant alleles in specific subgroups. The distribution of alleles DRB1*03, DRB1*04, DQB1*02, and DQB1*0302 was analyzed according to age of early onset (n = 30), late onset (n = 18) and gender. DRB1*03 (43.3% vs. 8.3%, pc < 10−3 ) and DQB1*02 (58.3% vs. 13.9%, pc < 10−3 ) phenotypes were more frequently expressed in the early onset disease group than in the late onset group. In contrast, the frequency of the DRB1*04 allele (16.7% vs. 47.2%, pc = 0.0013) and DQB1*0302 (13.3% vs. 47.2%, pc < 10−3 ) was more significant in the late onset disease group. This result reflects the strong linkage disequilibrium of these two pairs of HLA specificities. When patients were sub-grouped according to gender, a significant association in men with disease was found with DRB1*03 (32.7% vs. 7.7%, pc < 10−3 ) and DQB1*02 (57.7% vs. 16.3%, pc < 10−3 ); whereas the DRB1*04 (38.6% vs. 12.5%, pc < 10−3 ) and DQB1*0302 (34.1% vs. 10.4%, pc = 0.0016) phenotypes were more frequently expressed in women. Table 5 shows the frequency of the two most significant disease susceptibility haplotypes, DRB1*03/DQB1*02 and DRB1*04/DQB1*0302, according to age at onset and gender. While the DRB1*03/DQB1*02 haplotype was more frequently present in early onset MG patients (36.7%, pc = 0.0022), the DRB1*04/DQB1*0302 haplotype was more prevalent in late onset disease (33.3%, pc = 0.0099). A comparison between gender groups revealed that the frequency of DRB1*03/DQB1*02 was significantly increased in men with MG (28.8% vs. 7.7%, pc < 10−3 ) while the frequency of DRB1*04/DQB1*0302 was more profound in women patients (31.8% vs. 9.4%, pc < 10−3 ).
3. Results 4. Discussion 3.1. Clinical and immunological characteristics Table 1 summarizes the main clinical characteristics of the 48 unrelated Tunisian patients. The age at onset of disease ranged from 11 years to 76 years (mean 38.75 years). A cutoff age of 40 years was used to distinguish early onset (30 patients) from late onset (18 patients) of the disease. A serum anti-AChR antibody titer under 0.2 nmol/L was defined as negative.
MG is a heterogeneous disease that appears to occur in genetically susceptible individuals. Previously reported data associated this disease with various HLAs. In the present study, DRB and DQB allele distributions were investigated in Tunisian MG patients and compared with healthy controls. The genetics underlying the autoimmune response in MG is not well understood; although different HLA antigens have been
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N. Fekih-Mrissa et al. / Clinical Neurology and Neurosurgery 115 (2013) 32–36
Table 2 Allele frequency in myasthenia gravis patients and controls. (a) HLA-DRB1, 3, 4, 5 and (b) HLA-DQB1. HLA (a) HLA-DRB1 type DRB1*07 DRB1*04 DRB1*15 DRB1*03 DRB1*13 DRB1*11 DRB1*14 DRB1*09 (a) HLA-DRB3, 4, 5 types DRB3*01 DRB3*02 DRB4*01 DRB5*05 (b) HLA-DQB1 type DQB1*02 DQB1*0301 DQB1*0302 DQB1*05 DQB1*06
% MG patients (n = 48)
% Controls (n = 100)
P-value
14.6 28.12 10.4 30.2 6.25 8.33 1.04 1.04
18.0 14.5 10.0 11.5 16.0 16.0 6.0 0.5
0.46 0.005* 0.91 <10(−3 * 0.019 0.07 0.05 0.6
35.4 8.33 45.8 10.4
31.5 14.0 32.0 15.0
0.5 0.16 0.02 0.28
41.6 12.5 26.0 6.2 9.3
24.5 21.0 13.5 16.0 16.0
0.002* 0.08 0.007* 0.018 0.12
MG, myasthenia gravis; P-value, probability value; significant P-value is in bold, P < 0.05. * Corrected P value. Table 3 The frequencies of HLA DR/DQ allele haplotypes in the MG patients. HLA DR/DQ allele DRB1*/DQB1
% MG patients (n = 48)
% Controls (n = 100)
P-value
DRB1*07/DQB1*02 DRB1*04/DQB1*0302 DRB1*15/DQB1*06 DRB1*03/DQB1*02 DRB1*13/DQB1*06 DRB1*11/DQB1*0301 DRB1*13/DQB1*0301
10.4 19.7 6.2 26.0 3.1 5.2 3.1
14.5 8.0 8.0 9.0 7.0 7.0 9.0
0.21 0.004* 0.28 <10(−3 * 0.14 0.63 0.05
MG, myasthenia gravis; P-value, probability value; significant P-value is in bold, P < 0.05. * Corrected P value.
Table 4 Distribution of DRB1 and DQB1 alleles in MG subgroups according to age of disease onset (in MG group) and according to gender in MG group compared to healthy controls. Age at onset Allele DRB1*03 DRB1*04 DQB1*02 DQB1*0302
Male (%) <40 Y (n = 30) 43.3 16.7 58.3 13.3
(%) >40 Y (n = 18)
P-value −3 *
8.3 47.2 13.9 47.2
Female
% MG (n = 26)
<10( 0.0013* <10(−3 * <10(−3 *
32.7 19.2 57.7 19.2
% Control (n = 52)
P-value −3 *
7.7 17.3 16.3 16.3
<10( 0.77 10−3 * 0.65
% MG (n = 22)
% Control (n = 48)
27.3 38.6 22.7 34.1
15.6 12.5 33.3 10.4
P-value 0.10 <10(−3 * 0.2 0.0016
P-value, probability value; Y, years; significant P-value is in bold, P < 0.05. * Corrected P value.
frequently associated with disease in diverse ethnic populations [10,11]. Our study showed that the alleles DRB1*03, DRB1*04, DQB1*02, DQB1*03 were strongly associated with MG. This association was significant when considering patients as a whole and with subgrouping. However, the effect of DRB1*03 and DQB1*02 alleles was
particularly evident in the susceptibility to early onset MG and MG in men. Overall, without regard to gender or age of disease onset, our study demonstrated the strongest associations between the HLA-DR complex and MG were from DRB1*03 (30.2% vs. 11.5%, pc < 10−3 ) and DRB1*04 (28.12% vs. 14.5%, pc = 0.005) (Table 2).
Table 5 Distribution of DRB1/DQB1 haplotype in MG subgroups according to age of disease onset (in MG group) and according to gender in MG group compared to healthy controls. Age at onset
Male
Female
HLA DR DQ haplotypes
(%) <40 Y (n = 30)
(%) >40 Y (n = 18)
P-value
% MG (n = 26)
% Control (n = 52)
P-value
% MG (n = 22)
% Control (n = 48)
P-value
DRB1*03/DQB1*02 DRB1*04/DQB1*0302
36.7 11.7
8.33 33.3
0.0022* 0.0099*
28.8 9.6
7.7 6.7
<10(−3 * 0.52
22.7 31.8
9.4 9.4
0.032 <10(−3 *
P-value, probability value; Y, years; significant P-value is in bold, P < 0.05. * Corrected P value.
N. Fekih-Mrissa et al. / Clinical Neurology and Neurosurgery 115 (2013) 32–36
Similar results were previously found regarding the role of DRB1*03 to MG in Caucasian populations [7,8], and although DRB1*04 has never been reported to predispose for MG, it has been implicated in other autoimmune diseases [12]. Two alleles at the DQB1 locus, DQB1*02 and DQB1*0302, were also identified as susceptibility markers for MG (41.6% vs. 24.5%, pc = 0.002) and (26.0% vs. 13.5%, pc = 0.007), respectively. An association of MG with DQB1*02 was also observed in Caucasian, Venezuelan, Turkish, and Swedish patients [7,11,13,14]. DQB1*03 was implicated in MG among the Japanese population [15]. Heterogeneity of the disease is also evidenced by the observation that different alleles are associated with the disease depending on the various racial groups [16]. In our study, when all the MG patients were considered as a whole, the association was stronger with HLA haplotype DRB1*03/DQB1*02 (pc < 10−3 ) than with HLA DRB1*04/DQB1*0302 (pc = 0.004). The DRB1*03 and DQB1*02 alleles, as well as the DRB1*04 and DQB1*0302 alleles, are in linkage disequilibrium in both the Tunisian and other Mediterranean populations [17–21]. A strong association of DRB1 and DQB1 haplotypes in both the age of onset and gender disease subgroups was demonstrated. In our study, the haplotypes HLA DRB1*03/DQB1*02 and DRB1*04/DQB1*0302 were significantly associated with early onset MG (pc = 0.0022) and late onset MG (pc = 0.0099), respectively. Differences in frequency of HLA haplotype according to the age of onset have been found in other autoimmune diseases, such as juvenile rheumatoid arthritis and insulin-dependent diabetes mellitus [22–24]. A comparison between gender groups revealed an increased frequency of DRB1*03/DQB1*02 haplotype in men with MG when compared with that of healthy men (28.8% vs. 7.7%, pc < 10−3 ). Women, however, were more susceptible to the disease when bearing the DRB1*04/DQB1*0302 haplotype (31.8% vs. 9.4%, pc < 10−3 ). These findings provide further evidence that gender is a concomitant risk factor for this disease. Similarly, HLA association with MG was reported with the HLA DR3 haplotype in female patients with early onset disease [9]. In our study, the association between HLA types and other parameters such as thymoma, or anti-AChR antibody according to age of disease onset or gender was not performed. A larger group of patients would be required for a multivariate analysis of these relations. Our results concerning DRB1*03/DQB1*02 haplotype are in accord with other published results from around the world. Several studies conducted on Caucasians demonstrated the association of DRB1*03 and DQB1*02 with MG [25]. Additional studies also reported that HLA-DR3 and DQB1*02 were significantly and positively associated with MG [7,8], thereby suggesting that DQ2 is more important for susceptibility than HLA-DR3. Other studies showed that DRB3 and DQB1*02 were significantly more frequent among the MG subgroups [13]. However, a strong association with DR9 was reported in Chinese patients with juvenile onset MG [26], DRB1*16 and DRB1*13 were found to be associated with MG in Saudi patients [27]. The haplotype DRB1*04/DQB1*0302 has also been associated with autoimmune disease in Tunisians [28], in northern Europeans [29], and worldwide [24,30]. However, the authors believe that is the first research linking the haplotype DRB1*04/DQB1*0302 with MG disease. This study of Tunisian patients and controls determined two HLA-DR/DQ associations with MG. One part of this result was in accordance with previously published studies. However, we also revealed a new haplotype association, perhaps specific to our Tunisian patients. Further studies will be required to support the theory of MG susceptibility for those with these haplotype
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markers and, in particular, to specify this association in the Tunisian population. Acknowledgments We would like to thank Dr. Christian Winchell for his precious help in correcting this manuscript, and G. Bouaicha for her technical support. References [1] Engel AG. In: Myology AG, Engel BQ, Banker, editors. Acquired autoimmune myasthenia gravis. New York: McGraw-Hill Book Co.; 1986. p. 1925–54. [2] Calhoun RF, Ritter JH, Guthrie TJ, Pestronk A, Meyers BF, Patterson GA, et al. Results of transcervical thymectomy for myasthenia gravis in 100 consecutive patients. Annals of Surgery 1999;230:555–9. [3] Pirskanen R, Tiilikainen A, Hokkanen E. Histocompatibility (HL-A) antigens associated with myasthenia gravis. A preliminary report. Annals of Clinical Research 1972;4:304–6. [4] Fritze D, Hermann Jr C, Naeim F, Smith GS, Walford RL. HL-A antigens in myasthenia gravis. Lancet 1974;1:240–2. [5] Campbell RD, Milner CM. MHC genes in autoimmunity. Current Opinion in Immunology 1993;5:887–93. [6] Zelano G, Lino MM, Evoli A, Settesoldi D, Batocchi AP, Torrente I, et al. Tumor necrosis factor beta gene polymorphisms in myasthenia gravis. European Journal of Immunogenetics 1998;25:403–8. [7] Carlsson B, Wallin J, Pirskanen R, Matell G, Smith CI. Different HLA DR–DQ associations in subgroups of idiopathic myasthenia gravis. Immunogenetics 1990;31:285–90. [8] Vieira ML, Caillat-Zucman S, Gajdos P, Cohen-Kaminsky S, Casteur A, Bach JF. Identification by genomic typing of non-DR3 HLA class II genes associated with myasthenia gravis. Journal of Neuroimmunology 1993;47:115–22. [9] Compston DA, Vincent A, Newsom-Davis J, Batchelor JR. Clinical, pathological, HLA antigen and immunological evidence for disease heterogeneity in myasthenia gravis. Brain 1980;103:579–601. [10] Matsuki K, Juji T, Tokunaga K, Takamizawa M, Maeda H, Soda M, et al. HLA antigens in Japanese patients with myasthenia gravis. Journal of Clinical Investigation 1990;86:392–9. [11] Fernandez-Mestre MT, Vargas V, Montagnani S, Cotua M, Ogando V, Layrisse Z. HLA class II and class I polymorphism in Venezuelan patients with myasthenia gravis. Human Immunology 2004;65:54–9. [12] Matsushita S, Takahashi K, Motoki M, Komoriya K, Ikagawa S, Nishimura Y. Allele specificity of structural requirement for peptides bound to HLADRB1*0405 and -DRB1*0406 complexes: implication for the HLA-associated susceptibility to methimazole-induced insulin autoimmune syndrome. Journal of Experimental Medicine 1994;180:873–83. [13] Saruhan-Direskeneli G, Kilic¸ A, Parman Y, Serdaroglu P, Deymeer F. HLA-DQ polymorphism in Turkish patients with myasthenia gravis. Human Immunology 2006;67:352–8. [14] Hjelmström P, Giscombe R, Lefvert AK, Pirskanen R, Kockum I, Landin-Olsson M, et al. Polymorphic amino acid domains of the HLA-DQ molecule are associated with disease heterogeneity in myasthenia gravis. Journal of Neuroimmunology 1996;65:125–31. [15] Shinomiya N, Nomura Y, Segawa M. A variant of childhood-onset myasthenia gravis: HLA typing and clinical characteristics in Japan. Clinical Immunology 2004;110:154–8. [16] Christiansen FT, Pollack MS, Garlepp MJ, Dawkins RL. Myasthenia gravis and HLA antigens in American blacks and other races. Journal of Neuroimmunology 1984;7:121–9. [17] Ayed K, Ayed-Jendoubi S, Sfar I, Labonne MP, Gebuhrer L. HLA class-I and HLA class-II phenotypic, gene and haplotypic frequencies in Tunisians by using molecular typing data. Tissue Antigens 2004;64:520–32. [18] Hmida S, Gauthier A, Dridi A, Quillivic F, Genetet B, Boukef K, et al. HLA class II gene polymorphism in Tunisians. Tissue Antigens 1995;45:63–8. [19] Pédron B, Yakouben K, Guérin V, Borsali E, Auvrignon A, Landman J, et al. HLA alleles and haplotypes in French North African immigrants. Human Immunology 2006;67:540–50. [20] Brick C, Bennani N, Atouf O, Essakalli M. HLA-A, -B, -DR and -DQ allele and haplotype frequencies in the Moroccan population: a general population study. Transfusion Clinique et Biologique 2006;13:346–52. [21] Gibert M, Reviron D, Mercier P, Chiaroni J, Boetsch G. HLA-DRB1 and DQB1 polymorphisms in Southern France and genetic relationships with other Mediterranean populations. Human Immunology 2000;61:930–6. [22] Oen K, Schroeder M, Jacobson K, Anderson S, Wood S, Cheang M, et al. Juvenile rheumatoid arthritis in a Canadian First Nations (aboriginal) population: onset subtypes and HLA association. Journal of Rheumatology 1998;25: 783–90. [23] Sugihara S, Sakamaki T, Konda S, Murata A, Wataki K, Kobayashi Y, et al. Association of HLA-DR, DQ genotype with different beta-cell functions at IDDM diagnosis in Japanese children. Diabetes 1997;46:1893–7. [24] Izaabel H, Garchon HJ, Beaurain G, Biga M, Akhayat O, Bach JF, et al. Distribution of HLA class II alleles and haplotypes in insulin-dependent Moroccan diabetics. Human Immunology 1996;49:137–43.
36
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[25] Machens A, Loliger C, Pichlmeier U, Emskotter T, Busch C, Izbicki JR. Correlation of thymic pathology with HLA in myasthenia gravis. Clinical Immunology 1999;91:296–301. [26] Hawkins BR, Yu YL, Wong V, Woo E, Ip MS, Dawkins RL. Possible evidence for a variant of myasthenia gravis based on HLA and acetylcholine receptor antibody in Chinese patients. Quarterly Journal of Medicine 1989;70: 235–41. [27] Hajeer AH, Sawidan FA, Bohlega S, Saleh S, Sutton P, Shubaili A, et al. HLA class I and class II polymorphisms in Saudi patients with myasthenia gravis. International Journal of Immunogenetics 2009;36:169–72.
[28] Abid Kamoun H, Hmida S, Kaabi H, Abid A, Slimane Houissa H, Maamar M, et al. HLA polymorphism in type 1 diabetes Tunisians. Annales de Genetique 2002;45:45–50. [29] Thomson G, Robinson WP, Kuhner MK, Joe S, MacDonald MJ, Gottschall JL, et al. Genetic heterogeneity, modes of inheritance, and risk estimates for a joint study of Caucasians with insulin dependent diabetes mellitus. American Journal of Human Genetics 1988;43:799–816. [30] Marrosu MG, Murru M, Costa G, Murru R, Muntoni F, Cucca F. DRB1-DQA1DQB1 loci and multiple sclerosis predisposition in the Sardinian population. Human Molecular Genetics 1998;7:1235–7.
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Clinical Neurology and Neurosurgery journal homepage: www.elsevier.com/locate/clineuro
Association of HLA-DR/DQ polymorphism with myasthenia gravis in Tunisian patients Najiba Fekih-Mrissa a,∗ , Sarra Klai a , Jamel Zaouali b , Nasreddine Gritli a , Ridha Mrissa b a b
Laboratory of Molecular Biology, Department of Hematology, Military Hospital, Tunis, Tunisia Department of Neurology, Military Hospital, Tunis, Tunisia
a r t i c l e
i n f o
Article history: Received 25 May 2011 Received in revised form 16 January 2012 Accepted 1 April 2012 Available online 18 April 2012 Keywords: HLA class II polymorphism Myasthenia gravis
a b s t r a c t Background and objective: Myasthenia gravis (MG) is an autoimmune disorder of the neuromuscular junction. MG has been shown to be associated with many human leukocyte antigens (HLA) in different populations. The aim of this study was to investigate the probable association between HLA-DR/DQ alleles and MG in Tunisian patients. Patients and methods: HLA DR/DQ genotyping was performed using polymerase chain reaction sequencespecific primers (PCR-SSP) with 48 MG patients and 100 healthy individuals serving as the control group. Results: Myasthenia gravis in Tunisian patients was found to be associated with the following alleles (pc denotes Bonferroni corrected probability values): HLA-DRB1*03 (pc < 10−3 ), DRB1*04 (pc = 0.005), DQB1*02 (pc = 0.002) and, DQB1*03 (pc = 0.007). Conclusion: Our data demonstrated a new HLA-MG predisposition with DRB1*04. The DRB1*03, DRB1*04, DQB1*02, and DQB1*03 alleles also could be predisposing genetic factors for MG in the Tunisian population. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Myasthenia gravis (MG) is a rare and heterogeneous autoimmune disease characterized by progressive weakness and fatigue of the voluntary musculature. It is caused by immune-mediated loss of functioning acetylcholine receptors (AChR) from the post-synaptic membrane of the neuromuscular junction [1,2]. The association with human leukocyte antigens (HLA) has been known since the early 1970s [3,4]. These antigens strongly influence susceptibility to the development of MG [5,6]. In Caucasian populations, the most commonly cited alleles in the HLA complex that have been associated with MG are DRB1*03, DQB1*02, and DQB1*03 [7,8]. Studies of different populations have revealed that the frequencies of different HLA antigens vary depending on sex, age at disease onset, ethnic, and geographic origins of these populations [9].
Abbreviations: AChR, acetylcholine receptors; EMG, electromyography; HLA, human leukocyte antigens; LD, linkage disequilibrium; MG, myasthenia gravis; PCR, polymerase chain reaction; SSP, sequence-specific primer. ∗ Corresponding author at: Laboratory of Molecular Biology, Department of Hematology, Military Hospital. 1008 Mont Fleury, Tunis, Tunisia. Tel.: +216 22510488; fax: +216 70762084. E-mail address: [email protected] (N. Fekih-Mrissa). 0303-8467/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.clineuro.2012.04.001
In this study, we have genotyped HLA class II alleles in Tunisian MG patients and examined HLA class II association with MG subjects to understand HLA genotype effects on disease pathogenesis.
2. Methods 2.1. Subjects Forty-eight (22 women and 26 men) unrelated Tunisian MG patients were studied (male to female ratio = 1.18). The patients were referred from all regions of the country and were selected to have the generalized type of myasthenia; all other types were excluded. Myasthenia gravis was diagnosed according to conventional clinical criteria, electromyography, and/or a positive acetylcholine receptor antibodies test. The neostigmine test was positive for all patients. In AChR seronegative patients, the diagnosis was confirmed by electromyography and the positive neostigmine test. The main characteristics are summarized in Table 1. Serum anti-AChR antibody titer (nmol/L) was determined at the time of diagnosis using a conventional radioimmunoprecipitation assay. The controls consisted of 100 healthy unrelated individuals (48 women and 52 men) with diverse Tunisian origin similar to the patients (male to female ratio = 1.08). All participants gave informed consent before participation in the study.
N. Fekih-Mrissa et al. / Clinical Neurology and Neurosurgery 115 (2013) 32–36 Table 1 Characteristics of myasthenia gravis patients. Characteristics Sex Female Male Age at onset <40 Y >40 Y Thymoma EMGa (+) (−) Anti-AChr Abb (+) (−)
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3.2. HLA-DRB/DQB allele
Number of patients (n = 48)
Percentage
22 26
45.8 54.2
30 18 18
62.5 37.5 37.5
42 6
87.5 12.5
28 20
58.3 41.7
a EMG, electromyography on the extremity muscle; (+), myasthenic pattern positive, (−), myasthenic pattern negative.. b Anti-AChR Ab, anti-acetylcholine receptors antibody; (+), over 0.2 nm, (−), under 0.2 nm.
2.2. HLA-typing by DNA amplification Genomic DNA was extracted from peripheral blood samples of patients and healthy individuals using the QIAamp® DNA Blood Mini Kit (QIAGEN GmbH, Hilden, Germany). Low-resolution HLA typing was performed by PCR-SSP techniques according to Micro SSP DNA Typing Trays DRB/DQB (One Lambda Inc. Canoga Park, CA, USA). Amplified DNA fragments were detected by agarose gel electrophoresis (2.5% agarose gel), stained with ethidium bromide, and UV transillumination. One Lambda DNA/LMT software version 3.98 was used to detect specific DRB1 and DQB1 alleles.
2.3. Statistical analysis Allele frequencies were estimated by using the direct counting method. Haplotype frequencies were estimated using the EM algorithm and deviations from Hardy–Weinberg equilibrium were both performed using the Arlequin v.3.1 software (http://cmpg.unibe.ch/software/arlequin3). All analyses related to the case–control study were performed using the Statistical Package for the Social Sciences v.16 (SPSS Inc., Chicago, IL). Differences between cases and controls were evaluated by using the chisquare test or Fisher’s test for qualitative variables and t-test for quantitative variables. The odds ratios (OR) and 95% confidence intervals (CI) were also calculated. The Bonferroni method was used to adjust for type I errors due to multiple comparisons. Corrected probability values (pc ) were calculated for the number of comparisons made, i.e., the number of alleles tested. Probability values pc < 0.05 were considered statistically significant.
Table 2 (section a) shows the frequencies of DRB1, 3, 4 and 5 alleles in patients and controls. The prevalence of DRB1*03 and DRB1*04 was more elevated in MG patients than in controls (30.2% vs. 11.5%) and (28.12% vs. 14.5%). Both types DRB1*03 and DRB1*04 were positively and significantly associated with patients (pc < 10−3 , pc = 0.005 respectively). Both DRB3*01 and DRB4*01 were found to be in linkage disequilibrium (LD) with DRB1*03 and DRB1*04 respectively. The prevalence of types DRB3*01 and DRB4*01 in MG patients therefore can be explained by high frequencies of types DRB1*03 and DRB1*04, respectively. Table 2 (section b) shows the frequencies of the HLA DQB1 allele in patients and controls. Both DQB1*02 (41.6% vs. 24.5%, pc = 0.002) and DQB1*0302 (26.0% vs. 13.5%, pc = 0.007) were positively associated with MG. Table 3 shows the frequencies of HLA DR/DQ allele haplotypes in patients and control subjects. Only haplotypes exhibiting significant LD parameters between alleles were considered for study. The calculation of the LD parameter of two locus haplotypes, in both cases and controls, revealed that the most significant values of the LD parameters were for the pairs DRB1*03/DQB1*02 and DRB1*04/DQB1*0302. Compared with controls, patients showed a higher frequency of haplotypes DRB1*03/DQB1*02 (26.0% of patients vs. 9% of controls, pc < 10−3 ) and haplotype DRB1*04/DQB1*0302 (19.7% of patients vs. 8% of controls, pc = 0.004). Table 4 shows the frequency of significant alleles in specific subgroups. The distribution of alleles DRB1*03, DRB1*04, DQB1*02, and DQB1*0302 was analyzed according to age of early onset (n = 30), late onset (n = 18) and gender. DRB1*03 (43.3% vs. 8.3%, pc < 10−3 ) and DQB1*02 (58.3% vs. 13.9%, pc < 10−3 ) phenotypes were more frequently expressed in the early onset disease group than in the late onset group. In contrast, the frequency of the DRB1*04 allele (16.7% vs. 47.2%, pc = 0.0013) and DQB1*0302 (13.3% vs. 47.2%, pc < 10−3 ) was more significant in the late onset disease group. This result reflects the strong linkage disequilibrium of these two pairs of HLA specificities. When patients were sub-grouped according to gender, a significant association in men with disease was found with DRB1*03 (32.7% vs. 7.7%, pc < 10−3 ) and DQB1*02 (57.7% vs. 16.3%, pc < 10−3 ); whereas the DRB1*04 (38.6% vs. 12.5%, pc < 10−3 ) and DQB1*0302 (34.1% vs. 10.4%, pc = 0.0016) phenotypes were more frequently expressed in women. Table 5 shows the frequency of the two most significant disease susceptibility haplotypes, DRB1*03/DQB1*02 and DRB1*04/DQB1*0302, according to age at onset and gender. While the DRB1*03/DQB1*02 haplotype was more frequently present in early onset MG patients (36.7%, pc = 0.0022), the DRB1*04/DQB1*0302 haplotype was more prevalent in late onset disease (33.3%, pc = 0.0099). A comparison between gender groups revealed that the frequency of DRB1*03/DQB1*02 was significantly increased in men with MG (28.8% vs. 7.7%, pc < 10−3 ) while the frequency of DRB1*04/DQB1*0302 was more profound in women patients (31.8% vs. 9.4%, pc < 10−3 ).
3. Results 4. Discussion 3.1. Clinical and immunological characteristics Table 1 summarizes the main clinical characteristics of the 48 unrelated Tunisian patients. The age at onset of disease ranged from 11 years to 76 years (mean 38.75 years). A cutoff age of 40 years was used to distinguish early onset (30 patients) from late onset (18 patients) of the disease. A serum anti-AChR antibody titer under 0.2 nmol/L was defined as negative.
MG is a heterogeneous disease that appears to occur in genetically susceptible individuals. Previously reported data associated this disease with various HLAs. In the present study, DRB and DQB allele distributions were investigated in Tunisian MG patients and compared with healthy controls. The genetics underlying the autoimmune response in MG is not well understood; although different HLA antigens have been
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N. Fekih-Mrissa et al. / Clinical Neurology and Neurosurgery 115 (2013) 32–36
Table 2 Allele frequency in myasthenia gravis patients and controls. (a) HLA-DRB1, 3, 4, 5 and (b) HLA-DQB1. HLA (a) HLA-DRB1 type DRB1*07 DRB1*04 DRB1*15 DRB1*03 DRB1*13 DRB1*11 DRB1*14 DRB1*09 (a) HLA-DRB3, 4, 5 types DRB3*01 DRB3*02 DRB4*01 DRB5*05 (b) HLA-DQB1 type DQB1*02 DQB1*0301 DQB1*0302 DQB1*05 DQB1*06
% MG patients (n = 48)
% Controls (n = 100)
P-value
14.6 28.12 10.4 30.2 6.25 8.33 1.04 1.04
18.0 14.5 10.0 11.5 16.0 16.0 6.0 0.5
0.46 0.005* 0.91 <10(−3 * 0.019 0.07 0.05 0.6
35.4 8.33 45.8 10.4
31.5 14.0 32.0 15.0
0.5 0.16 0.02 0.28
41.6 12.5 26.0 6.2 9.3
24.5 21.0 13.5 16.0 16.0
0.002* 0.08 0.007* 0.018 0.12
MG, myasthenia gravis; P-value, probability value; significant P-value is in bold, P < 0.05. * Corrected P value. Table 3 The frequencies of HLA DR/DQ allele haplotypes in the MG patients. HLA DR/DQ allele DRB1*/DQB1
% MG patients (n = 48)
% Controls (n = 100)
P-value
DRB1*07/DQB1*02 DRB1*04/DQB1*0302 DRB1*15/DQB1*06 DRB1*03/DQB1*02 DRB1*13/DQB1*06 DRB1*11/DQB1*0301 DRB1*13/DQB1*0301
10.4 19.7 6.2 26.0 3.1 5.2 3.1
14.5 8.0 8.0 9.0 7.0 7.0 9.0
0.21 0.004* 0.28 <10(−3 * 0.14 0.63 0.05
MG, myasthenia gravis; P-value, probability value; significant P-value is in bold, P < 0.05. * Corrected P value.
Table 4 Distribution of DRB1 and DQB1 alleles in MG subgroups according to age of disease onset (in MG group) and according to gender in MG group compared to healthy controls. Age at onset Allele DRB1*03 DRB1*04 DQB1*02 DQB1*0302
Male (%) <40 Y (n = 30) 43.3 16.7 58.3 13.3
(%) >40 Y (n = 18)
P-value −3 *
8.3 47.2 13.9 47.2
Female
% MG (n = 26)
<10( 0.0013* <10(−3 * <10(−3 *
32.7 19.2 57.7 19.2
% Control (n = 52)
P-value −3 *
7.7 17.3 16.3 16.3
<10( 0.77 10−3 * 0.65
% MG (n = 22)
% Control (n = 48)
27.3 38.6 22.7 34.1
15.6 12.5 33.3 10.4
P-value 0.10 <10(−3 * 0.2 0.0016
P-value, probability value; Y, years; significant P-value is in bold, P < 0.05. * Corrected P value.
frequently associated with disease in diverse ethnic populations [10,11]. Our study showed that the alleles DRB1*03, DRB1*04, DQB1*02, DQB1*03 were strongly associated with MG. This association was significant when considering patients as a whole and with subgrouping. However, the effect of DRB1*03 and DQB1*02 alleles was
particularly evident in the susceptibility to early onset MG and MG in men. Overall, without regard to gender or age of disease onset, our study demonstrated the strongest associations between the HLA-DR complex and MG were from DRB1*03 (30.2% vs. 11.5%, pc < 10−3 ) and DRB1*04 (28.12% vs. 14.5%, pc = 0.005) (Table 2).
Table 5 Distribution of DRB1/DQB1 haplotype in MG subgroups according to age of disease onset (in MG group) and according to gender in MG group compared to healthy controls. Age at onset
Male
Female
HLA DR DQ haplotypes
(%) <40 Y (n = 30)
(%) >40 Y (n = 18)
P-value
% MG (n = 26)
% Control (n = 52)
P-value
% MG (n = 22)
% Control (n = 48)
P-value
DRB1*03/DQB1*02 DRB1*04/DQB1*0302
36.7 11.7
8.33 33.3
0.0022* 0.0099*
28.8 9.6
7.7 6.7
<10(−3 * 0.52
22.7 31.8
9.4 9.4
0.032 <10(−3 *
P-value, probability value; Y, years; significant P-value is in bold, P < 0.05. * Corrected P value.
N. Fekih-Mrissa et al. / Clinical Neurology and Neurosurgery 115 (2013) 32–36
Similar results were previously found regarding the role of DRB1*03 to MG in Caucasian populations [7,8], and although DRB1*04 has never been reported to predispose for MG, it has been implicated in other autoimmune diseases [12]. Two alleles at the DQB1 locus, DQB1*02 and DQB1*0302, were also identified as susceptibility markers for MG (41.6% vs. 24.5%, pc = 0.002) and (26.0% vs. 13.5%, pc = 0.007), respectively. An association of MG with DQB1*02 was also observed in Caucasian, Venezuelan, Turkish, and Swedish patients [7,11,13,14]. DQB1*03 was implicated in MG among the Japanese population [15]. Heterogeneity of the disease is also evidenced by the observation that different alleles are associated with the disease depending on the various racial groups [16]. In our study, when all the MG patients were considered as a whole, the association was stronger with HLA haplotype DRB1*03/DQB1*02 (pc < 10−3 ) than with HLA DRB1*04/DQB1*0302 (pc = 0.004). The DRB1*03 and DQB1*02 alleles, as well as the DRB1*04 and DQB1*0302 alleles, are in linkage disequilibrium in both the Tunisian and other Mediterranean populations [17–21]. A strong association of DRB1 and DQB1 haplotypes in both the age of onset and gender disease subgroups was demonstrated. In our study, the haplotypes HLA DRB1*03/DQB1*02 and DRB1*04/DQB1*0302 were significantly associated with early onset MG (pc = 0.0022) and late onset MG (pc = 0.0099), respectively. Differences in frequency of HLA haplotype according to the age of onset have been found in other autoimmune diseases, such as juvenile rheumatoid arthritis and insulin-dependent diabetes mellitus [22–24]. A comparison between gender groups revealed an increased frequency of DRB1*03/DQB1*02 haplotype in men with MG when compared with that of healthy men (28.8% vs. 7.7%, pc < 10−3 ). Women, however, were more susceptible to the disease when bearing the DRB1*04/DQB1*0302 haplotype (31.8% vs. 9.4%, pc < 10−3 ). These findings provide further evidence that gender is a concomitant risk factor for this disease. Similarly, HLA association with MG was reported with the HLA DR3 haplotype in female patients with early onset disease [9]. In our study, the association between HLA types and other parameters such as thymoma, or anti-AChR antibody according to age of disease onset or gender was not performed. A larger group of patients would be required for a multivariate analysis of these relations. Our results concerning DRB1*03/DQB1*02 haplotype are in accord with other published results from around the world. Several studies conducted on Caucasians demonstrated the association of DRB1*03 and DQB1*02 with MG [25]. Additional studies also reported that HLA-DR3 and DQB1*02 were significantly and positively associated with MG [7,8], thereby suggesting that DQ2 is more important for susceptibility than HLA-DR3. Other studies showed that DRB3 and DQB1*02 were significantly more frequent among the MG subgroups [13]. However, a strong association with DR9 was reported in Chinese patients with juvenile onset MG [26], DRB1*16 and DRB1*13 were found to be associated with MG in Saudi patients [27]. The haplotype DRB1*04/DQB1*0302 has also been associated with autoimmune disease in Tunisians [28], in northern Europeans [29], and worldwide [24,30]. However, the authors believe that is the first research linking the haplotype DRB1*04/DQB1*0302 with MG disease. This study of Tunisian patients and controls determined two HLA-DR/DQ associations with MG. One part of this result was in accordance with previously published studies. However, we also revealed a new haplotype association, perhaps specific to our Tunisian patients. Further studies will be required to support the theory of MG susceptibility for those with these haplotype
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markers and, in particular, to specify this association in the Tunisian population. Acknowledgments We would like to thank Dr. Christian Winchell for his precious help in correcting this manuscript, and G. Bouaicha for her technical support. References [1] Engel AG. In: Myology AG, Engel BQ, Banker, editors. Acquired autoimmune myasthenia gravis. New York: McGraw-Hill Book Co.; 1986. p. 1925–54. [2] Calhoun RF, Ritter JH, Guthrie TJ, Pestronk A, Meyers BF, Patterson GA, et al. Results of transcervical thymectomy for myasthenia gravis in 100 consecutive patients. Annals of Surgery 1999;230:555–9. [3] Pirskanen R, Tiilikainen A, Hokkanen E. Histocompatibility (HL-A) antigens associated with myasthenia gravis. A preliminary report. Annals of Clinical Research 1972;4:304–6. [4] Fritze D, Hermann Jr C, Naeim F, Smith GS, Walford RL. HL-A antigens in myasthenia gravis. Lancet 1974;1:240–2. [5] Campbell RD, Milner CM. MHC genes in autoimmunity. Current Opinion in Immunology 1993;5:887–93. [6] Zelano G, Lino MM, Evoli A, Settesoldi D, Batocchi AP, Torrente I, et al. Tumor necrosis factor beta gene polymorphisms in myasthenia gravis. European Journal of Immunogenetics 1998;25:403–8. [7] Carlsson B, Wallin J, Pirskanen R, Matell G, Smith CI. Different HLA DR–DQ associations in subgroups of idiopathic myasthenia gravis. Immunogenetics 1990;31:285–90. [8] Vieira ML, Caillat-Zucman S, Gajdos P, Cohen-Kaminsky S, Casteur A, Bach JF. Identification by genomic typing of non-DR3 HLA class II genes associated with myasthenia gravis. Journal of Neuroimmunology 1993;47:115–22. [9] Compston DA, Vincent A, Newsom-Davis J, Batchelor JR. Clinical, pathological, HLA antigen and immunological evidence for disease heterogeneity in myasthenia gravis. Brain 1980;103:579–601. [10] Matsuki K, Juji T, Tokunaga K, Takamizawa M, Maeda H, Soda M, et al. HLA antigens in Japanese patients with myasthenia gravis. Journal of Clinical Investigation 1990;86:392–9. [11] Fernandez-Mestre MT, Vargas V, Montagnani S, Cotua M, Ogando V, Layrisse Z. HLA class II and class I polymorphism in Venezuelan patients with myasthenia gravis. Human Immunology 2004;65:54–9. [12] Matsushita S, Takahashi K, Motoki M, Komoriya K, Ikagawa S, Nishimura Y. Allele specificity of structural requirement for peptides bound to HLADRB1*0405 and -DRB1*0406 complexes: implication for the HLA-associated susceptibility to methimazole-induced insulin autoimmune syndrome. Journal of Experimental Medicine 1994;180:873–83. [13] Saruhan-Direskeneli G, Kilic¸ A, Parman Y, Serdaroglu P, Deymeer F. HLA-DQ polymorphism in Turkish patients with myasthenia gravis. Human Immunology 2006;67:352–8. [14] Hjelmström P, Giscombe R, Lefvert AK, Pirskanen R, Kockum I, Landin-Olsson M, et al. Polymorphic amino acid domains of the HLA-DQ molecule are associated with disease heterogeneity in myasthenia gravis. Journal of Neuroimmunology 1996;65:125–31. [15] Shinomiya N, Nomura Y, Segawa M. A variant of childhood-onset myasthenia gravis: HLA typing and clinical characteristics in Japan. Clinical Immunology 2004;110:154–8. [16] Christiansen FT, Pollack MS, Garlepp MJ, Dawkins RL. Myasthenia gravis and HLA antigens in American blacks and other races. Journal of Neuroimmunology 1984;7:121–9. [17] Ayed K, Ayed-Jendoubi S, Sfar I, Labonne MP, Gebuhrer L. HLA class-I and HLA class-II phenotypic, gene and haplotypic frequencies in Tunisians by using molecular typing data. Tissue Antigens 2004;64:520–32. [18] Hmida S, Gauthier A, Dridi A, Quillivic F, Genetet B, Boukef K, et al. HLA class II gene polymorphism in Tunisians. Tissue Antigens 1995;45:63–8. [19] Pédron B, Yakouben K, Guérin V, Borsali E, Auvrignon A, Landman J, et al. HLA alleles and haplotypes in French North African immigrants. Human Immunology 2006;67:540–50. [20] Brick C, Bennani N, Atouf O, Essakalli M. HLA-A, -B, -DR and -DQ allele and haplotype frequencies in the Moroccan population: a general population study. Transfusion Clinique et Biologique 2006;13:346–52. [21] Gibert M, Reviron D, Mercier P, Chiaroni J, Boetsch G. HLA-DRB1 and DQB1 polymorphisms in Southern France and genetic relationships with other Mediterranean populations. Human Immunology 2000;61:930–6. [22] Oen K, Schroeder M, Jacobson K, Anderson S, Wood S, Cheang M, et al. Juvenile rheumatoid arthritis in a Canadian First Nations (aboriginal) population: onset subtypes and HLA association. Journal of Rheumatology 1998;25: 783–90. [23] Sugihara S, Sakamaki T, Konda S, Murata A, Wataki K, Kobayashi Y, et al. Association of HLA-DR, DQ genotype with different beta-cell functions at IDDM diagnosis in Japanese children. Diabetes 1997;46:1893–7. [24] Izaabel H, Garchon HJ, Beaurain G, Biga M, Akhayat O, Bach JF, et al. Distribution of HLA class II alleles and haplotypes in insulin-dependent Moroccan diabetics. Human Immunology 1996;49:137–43.
36
N. Fekih-Mrissa et al. / Clinical Neurology and Neurosurgery 115 (2013) 32–36
[25] Machens A, Loliger C, Pichlmeier U, Emskotter T, Busch C, Izbicki JR. Correlation of thymic pathology with HLA in myasthenia gravis. Clinical Immunology 1999;91:296–301. [26] Hawkins BR, Yu YL, Wong V, Woo E, Ip MS, Dawkins RL. Possible evidence for a variant of myasthenia gravis based on HLA and acetylcholine receptor antibody in Chinese patients. Quarterly Journal of Medicine 1989;70: 235–41. [27] Hajeer AH, Sawidan FA, Bohlega S, Saleh S, Sutton P, Shubaili A, et al. HLA class I and class II polymorphisms in Saudi patients with myasthenia gravis. International Journal of Immunogenetics 2009;36:169–72.
[28] Abid Kamoun H, Hmida S, Kaabi H, Abid A, Slimane Houissa H, Maamar M, et al. HLA polymorphism in type 1 diabetes Tunisians. Annales de Genetique 2002;45:45–50. [29] Thomson G, Robinson WP, Kuhner MK, Joe S, MacDonald MJ, Gottschall JL, et al. Genetic heterogeneity, modes of inheritance, and risk estimates for a joint study of Caucasians with insulin dependent diabetes mellitus. American Journal of Human Genetics 1988;43:799–816. [30] Marrosu MG, Murru M, Costa G, Murru R, Muntoni F, Cucca F. DRB1-DQA1DQB1 loci and multiple sclerosis predisposition in the Sardinian population. Human Molecular Genetics 1998;7:1235–7.