HLA Class II and class I polymorphism in venezuelan patients with myasthenia gravis

HLA Class II and Class I Polymorphism in Venezuelan Patients With Myasthenia Gravis Mercedes T. Ferna´ndez-Mestre, Vivian Vargas, Silvia Montagnani, Maritza Cotu´a, Violeta Ogando, and Zulay Layrisse ABSTRACT: Oligotyping performed among ethnically mixed Venezuelan patients with myasthenia gravis (MG) and controls has revealed positive associations of HLA class I A*31, B*08, B*39, B*40, C*15, C*17, and class II DRB1*09 and negative associations of DQB1*06 and DQA1*02 with the disease. Sequential removal of human leukocyte antigen B (HLA-B) alleles when relative predispositional effects (RPEs) were looked for demonstrated that B*08 is the allele group with the largest contribution in the overall MG patients followed by B*39 and B*40. Several specificities (A*31, B*08, C*17, DRB1*03, DQA1*05, and DQB1*02) indicated increased frequencies among patients with thymic hyperplasia versus patients without hyperplasia or controls. Tests to identify alleles with the strongest association to MG in our patients detected ABBREVIATIONS AChR acetylcholine receptor HLA human leukocyte antigen MG myasthenia gravis MHC major histocompatibility complex

INTRODUCTION Myasthenia gravis (MG) is a heterogeneous autoimmune disease characterized by weakness and fatigability of skeletal muscle [1] Autoantibodies and reactive T cells directed at the acetylcholine receptor (AChR) of the From the Centro de Medicina Experimental “Miguel Layrisse” (M.T.F.M., S.M., V.O., Z.L.), Instituto Venezolano de Investigaciones Cientı´ficas, and the Servicio de Neurologı´a (V.V., M.C.), Hospital Universitario de Caracas, Caracas, Venezuela. Mercedes T. Ferna´ndez-Mestre and Zulay Layrisse contributed equally to this manuscript. Address reprint requests to: Dr. Layrisse, Centro de Medicina Experimental “Miguel Layrisse,” Instituto Venezolano de Investigaciones Cientificas, Km 11 Carretera Panamericana, Apt. 21827, Caracas 1020 A, Venezuela; Tel: ⫹58 (212) 5041155; Fax: ⫹58 (212) 5041086; E-mail: [email protected]. Received June 27, 2003; revised October 3, 2003; accepted October 7, 2003. Human Immunology 65, 54 –59 (2004) © American Society for Histocompatibility and Immunogenetics, 2004 Published by Elsevier Inc.

DRB1*13 and B*38 as possible predisposing secondarily associated alleles in patients with hyperplasia. The associations observed disappear after Bonferoni correction of probability values and have been described in patients of Caucasian and/or Oriental ethnic background. Thus, our results reflect the heterogeneity of our population and of the patients tested and suggest a limited influence of several HLA genes in this heterogenous disease or that these might be only markers of nearby non-HLA genes responsible for the susceptibility or resistance effect. Human Immunology 65, 54 –59 (2004). © American Society for Histocompatibility and Immunogenetics, 2004. Published by Elsevier Inc. KEYWORDS: HLA; myasthenia gravis; oligotyping; polymorphism

PCR SSOP SSP

polymerase chain reaction sequence-specific oligonucleotide probes sequence-specific primer

neuromuscular junction are responsible for a decrease of AChR receptors in MG [2]. Thymic abnormalities, such as hyperplasia or thymoma, are frequently present in these patients, suggesting a role for the thymus in pathogenesis. The presence of associated autoimmune disorders in MG patients and a positive response to various immunomodulating therapies, including plasmapheresis, corticosteroids, intravenous immunoglobulin, other immunosuppressants and thymectomy, have demonstrated that autoimmunizing mechanisms play important roles in the pathophysiology of the disease [3]. Immunogenetic studies in healthy and diseased populations have demonstrated that several genes besides major histocompatibility complex (MHC) genes play an 0198-8859/04/$–see front matter doi:10.1016/j.humimm.2003.10.003

HLA Polymorphisms in Venezuelan Patients With Myasthenia Gravis

important role in determining an individual’s susceptibility to the development of MG [4, 5]. The association with human leukocyte antigen (HLA) antigens has been known since the early 1970s [6, 7]. However, the nature and the intensity of the association vary depending on sex, age at disease onset, and thymic histology of the patients [8], and differ between Caucasian and Oriental populations [9, 10]. In this study we have analyzed HLA class I and class II polymorphisms in an ethnically mixed group of Venezuelan MG patients as a contribution to understand HLA genotype effects on disease pathogenesis. MATERIALS AND METHODS Patients Forty-nine unrelated Venezuelan MG patients were studied. The patients were referred from all regions of the country to the outpatient Neurological Service of our largest University Hospital (Universidad Central de Venezuela). They were selected in order to have patients with antecedents of Venezuelan ancestors of at least three generations and without family history of autoimmune diseases. Patients and controls are Venezuelan mestizos, that is to say, descendants of different degrees of admixture between European migrants (Spain, Portugal, Italy, Germany, etc) with descendants of local Amerindians and African slaves, who constitute the Venezuelan extant hybrid population. Generalized MG was diagnosed according to conventional clinical and electrophysiologic criteria, however, AChR antibody concentration in serum was not measured. The patients were classified into different clinical groups based on the extent and progression of muscle involvement [11]. Mild generalized weakness (IIA) and moderate generalized weakness (IIB) were encountered in 41% and 59% of patients, respectively. Histologic data of the gland was provided following thymectomy in 35 patients (29 females and 6 males); nine patients were not thymectomized (Table 1). One hundred and sixty unrelated healthy individuals with ethnic background similar to that of the patients were tested as controls. HLA Typing Genomic DNA was extracted from peripheral blood using the salting-out method [12]. The second exon of the DRB1, DQA1, and DQB1 genes was separately amplified by the polymerase chain reaction (PCR) using protocols, primer pairs, and probes selected for the 12th International Histocompatibility Workshop [13]. Amplified DNA was denatured, blotted, and immobilized by ultraviolet radiation onto nylon membranes. Membranes were hybridized with nonradioactively labeled HLA-DRB1, DQA1, and DQB1 sequence-specific oli-

55

TABLE 1 Clinical and histologic characteristics of myasthenia gravis patients Number of patients (n ⫽ 49) Gender Thymic histology Thymoma Hyperplasia Neither thymoma nor hyperplasia Unknown Not thymectomized Total patients

Male

Female

Age at disease onset ⬍40 years old

⬎40 years old

1 1 4

6 16 7

5 15 9

2 2 2

1 1

4 8

3 5

2 4

8 (16%)

41 (84%)

37 (76%)

12 (24%)

Early onset (⬍40 years old); late onset (⬎40 years old).

gonucleotide probes (SSOP). A chemiluminescence substrate was used for detection according to the manufacturer’s protocol (Boehringer Mannheim Biochemical, Mannheim, Germany). DNAs from cell lines with known HLA class II alleles were used as controls for hybridization. HLA class I typing was carried out using a sequence-specific primer (SSP) test (One Lambda, Inc., Los Angeles CA, USA). After the PCR process, the amplified DNA fragments were separated by agarose gel electrophoresis and visualized with ethidium bromide and exposure to ultraviolet light. Interpretation of the PCR-SSP result was based on the presence or absence of a specific amplified DNA fragment. Statistical Analysis HLA class II phenotypes were assigned to each individual based on the oligotyping results applying the Score software (13th IHW), and for class I using an HLA software from One Lambda. HLA-DRB1, DQA1, DQB1, A, B, and C low resolution specificities and haplotype frequencies were determined by maximum likelihood using the Arlequin computer program (University of Geneva, Geneva, Switzerland). The statistical significance of allele frequency differences between patients and controls was estimated by Fisher’s exact test; p values were corrected multiplying by the number of comparisons made and were considered significant when p ⬍ 0.05. Relative risk were calculated as odds ratios (OR) according to Woolf’s formula [14] or by the modified method described by Haldane [15] when one element of the equation was zero. To detect the strongest association between pairs of alleles Svejgaard’s method [16] was applied using 2 ⫻ 4 tables giving the four phenotypic combinations in patients and controls. The relative predispositional effect (RPEs) [17] of the predisposing or protective HLA alleles was also applied to our data in order to identify associ-

56

M.T. Ferna´ ndez-Mestre et al.

TABLE 2 HLA class II and class I specificities frequency differences among MG patients (entire group) compared with controls, among MG patients with or without hyperplasia, and among MG patients with thymic hyperplasia and controls MG vs controls

DRB1*03 *09 DQB1*02 *06 DQA1*02 *05

HLA-A*02 *31 HLA-B*08 *39 *40 HLA-C*15 *17

Controls (n ⫽ 160)

Total MG patients (n ⫽ 49)

12 1.9 25 30 18 44

20 10 29 17 4 37

Controls (n ⫽ 49)

Total MG patients (n ⫽ 41)

67 4 6 8 12 10 4

51 17 22 24 29 32 20

MG patients

Controls vs MG

OR

Without hyperplasia (n ⫽ 18)

With Hyperplasia (n ⫽ 17)

1.9 5.94† 1.2 0.46† 0.21† 0.75

17 18 17 17 6 20

41 6 53 29 6 75

OR

Controls (n ⫽ 160)

With Hyperplasia (n ⫽ 17)

3.5 0.3 5.6† 2.08 1.00 6.6†

12 1.9 25 30 18 44

41 6 53 29 6 75 With hyperplasia (n ⫽ 16) 31 25 31 25 13 13 31

OR

Without hyperplasia (n ⫽ 13)

With hyperplasia (n ⫽ 16)

OR

Controls (n ⫽ 160)

0.51 4.8† 4.3† 3.6† 2.97† 4.2† 5.7†

62 15 23 31 23 39 23

31 25 31 25 13 13 31

0.28 1.83 1.51 0.75 0.47 0.22 1.51

67 4 6 8 12 10 4

OR 5.19† 3.27 3.29 3.85

OR 0.48† 1.83† 6.96†

1.51†

p ⬍ 0.05; pc ⫽ not significant. Results are presented as phenotype frequency percent. Abbreviations: HLA ⫽ human leukocyte antigen; MG ⫽ myasthenia gravis; OR ⫽ odds ratio. †

ations sequentially according to their strength. Due to the small number of patients it was not possible to obtain ␹2 values in all, and significance was obtained using Fisher’s exact tests. RESULTS HLA Class II Prevalences in MG Patients Overall or When Grouped According to Thymic Pathology When the HLA-DRB1, -DQA1, and -DQB1 alleles were considered among the whole series of MG patients, there was a significant increase of DRB1*09 (OR ⫽ 5.94). Conversely, DQB1*06 (OR ⫽ 0.46) and DQA1*02 (OR ⫽ 0.21) indicated decreases. All of these probability values (pc) lost significance after correction (Table 2). The well-known associations of thymic pathology with sex and HLA were also evident. As expected, patients with thymic hyperplasia are almost all female with early MG onset: the increases in DRB1*03 (OR ⫽ 3.5), DQB1*02 (OR ⫽ 5.6), and/or DQA1*05 (OR ⫽ 6.6) were largely restricted to the patients with thymic hyperplasia. The frequency differences are statistically significant for DQB1*02 and DQA1*05, although not after correction of the p value.

HLA Class I Prevalences in MG Patients Overall or When Grouped According to Thymic Pathology HLA-A, -B, and -C specificities were determined in patients and in a subgroup of the controls. HLA-A*31, -B*08, -B*39, -B*40, -C*15, and -C*17 were increased among patients, whereas B*15 and C*12 were decreased (data not shown); again, these differences lost significance after correction of the p values (Table 2). The increases in A*31 (OR ⫽ 1.83), B*08 (OR ⫽ 6.96), and C*17 (OR ⫽ 1.51) appear greater among patients with thymic hyperplasia, unlike those in B*39, B*40, and C*15. Interestingly, A*02 was decreased only in patients with hyperplasia. MG Patients With Thymoma The seven MG patients with thymoma manifested increases in B*39 (OR ⫽ 12.86), C*17 (OR ⫽ 5.17), and DRB1*09 (OR ⫽ 5.2) relative to the other patients, although none of them had DRB1*03 or DQA1*05 (data not shown). HLA Haplotypes Among MG Patients and Controls The most frequent two-loci haplotypes among MG patients are illustrated in Table 3. It can be seen that

HLA Polymorphisms in Venezuelan Patients With Myasthenia Gravis

57

TABLE 3 Most frequent two-loci haplotypes in MG patients and controls HLA

MG patients (HF)

Control patients (HF)

A*02 B*40 A*01 B*08 A*02 B*39 B*40 C*15 B*08 C*07 B*35 C*04 DRB1*03 A*01 DRB1*03 B*08 DRB1*03 C*07 DRB1*11 C*03 DRB1*04 DQB1*03 DRB1*03 DQB1*02 DRB1*11 DQB1*03 DRB1*01 DQB1*05 DRB1*02 DQB1*03 DRB1*02 DQA1*01 DQB1*03 DQA1*03 DQB1*05 DQA1*01 DQB1*03 DQA1*01

0.0732 0.0488 0.0489 0.0854 0.0732 0.0610 0.0609 0.0731 0.0731 0.0731 0.1633 0.0816 0.0714 0.0612 0.0612 0.0816 0.1837 0.1735 0.0612

0.0306 0.0204 0 0 0.0204 0.0306 0.011 0.022 0.022 0.0113 0.170 0.068 0.1136 0.022 0.011 0.0454 0.1590 0.0681 0

OR

p Value

pc Value

11.88 21.52

0.039 0.00301

NS NS

7.37

0.0446

NS

3.36 13.29

0.0164 0.01835

NS NS

Abbreviations: HF ⫽ haplotype frequencies (calculated by maximum likelihood methods); MG ⫽ myasthenio gravis; NS ⫽ not significant; OR ⫽ odd ratios; p ⫽ probability; pc ⫽ probability corrected.

haplotypes A*01 DRB1*03; B*08 DRB1*03; C*07 DRB1*03; A*01 B*08; and B*08 C*07 are present with increased frequency in the total group of MG patients. These combinations have been reported with high frequency among healthy and diseased Caucasoid populations [18, 19]. Detection of the Strongest Association Because several alleles were found positively or negatively associated to MG in our patients, the independent effect of each of the following pairs of associated alleles was analyzed following Svejgaard [16]: A31–B39; B40 – C15; B8 –DR9; B8 –DR3; DR3–DQB1*02; DR3– DQA1*05; DQB1*02–DQA1*05. Only DRB1*03 and DQB1*02 were found significantly associated (pc ⬍ 0.0001) in the overall group of patients and controls. Although only among the patients with hyperplasia, B8 and DR3 were found significantly associated (pc ⫽ 0.01; data not shown). The relative predispositional effect [17] was tested separately for alleles at each locus. In the overall patients group, when patients and controls carrying DRB1*09 were removed, no secondary predisposing or protective associated allele was found. Removal of DQA1*05 or DQB1*06 revealed no other DQ associated alleles. Analysis of class I alleles by locus indicated no other locus A associated besides A31. At the B locus, a decreasing association strength for B*08, B*39, and B*40 was seen. At the C locus, the predisposing effect of C*17 remained (OR ⫽ 5.6; p ⫽ 0.02). None of the p values maintained significance after Bonferroni’s correction (Table 4).

Among the patients with hyperplasia removal of DR3⫹ patients and controls disclosed a weak association (OR ⫽ 3.0, p ⫽ 0.035) with DR13, which disappeared after correction. Removal of patients and controls A*31⫹, depicted a weak protective effect of A*02 (OR ⫽ 0.036, p ⫽ 0.039). Interestingly, removal of B*08 demonstrated a protective effect of B*38 (RR ⫽ 18.7; p ⫽ 0.048; Table 4). DISCUSSION The genetics of the autoimmune response in MG are not well understood, although associations with HLA, tumor necrosis factor, and T-cell receptor V␤ have been reported [5, 20, 21]. However, one important feature in MG is the consistent association with different HLA antigens in populations with different ethnic background [9, 10]. The results of our study among MG patients of mixed ethnic origin have illustrated several positive and negative associations, suggesting predisposing or protective effects of MHC genes on the development of the disease. Although nonsignificant after correction of the p value, an association was observed in the total group of patients with DRB1*09, DQB1*06, and DQA1*02. These findings are interesting and could be explained by the ethnic admixture existing in the Venezuelan population. It is important to emphasize that the populations living in the American continent are highly admixed due to continuous population migrations from other continents and within the continent [19, 22].

58

M.T. Ferna´ ndez-Mestre et al.

TABLE 4 Selected relative predispositional effects of HLA allele groups in overall MG patients and in MG patients with hyperplasia. 1st Round of comparison Allele

Obs.

Overall MG patients: B*08 9 B*39 10 B*40 12

2nd Round B*08 removed

Exp.

␹2

OR

p Value

Exp.

␹2

OR

p Value

Exp.

␹2

OR

p Value

2.51 4 6

3.3 3.04 2.7

3.9 3.26 2.63

0.03 0.04 0.05

— 3.07 4.61

— 3.7 3.4

— 3.61 2.92

— 0.027 0.032

— — 4.15

4.45

3.3

0.02

1st Round of comparison C*15 C*17

13 8

4.65 1.80

4.11 ND

3.32 4.92

2nd Round C*15 removed 0.02 0.03

— 1.60

1st Round of comparison Allele

Obs.

Exp.

MG patients with hyperplasia A*31 4 6.4 A*02 5 10.77 A*01 6 2.46

5 2

1 0

7 6

2 2.76

— 5.64

— 0.02

␹2

OR

p Value

Exp.

␹2

OR

p Value

ND 2.97 ND

6.86 0.37 3.00

0.03 0.04 NS

— 9.6 1.97

— 2.06 ND

— 0.415 3.47

— NS 0.04

ND ND

3.90 8.67

2nd Round B*08 removed 0.02 n.s.

— 0

1st Round of comparison DR*03 DR*13

— ND

3rd Round

2nd Round A*31 removed

1st Round of comparison B*08 B*38

3rd Round B*39 removed

ND ND

4.11 2.42

— n.d.

— 18.7

— 3.2

— ND

— 3.02

Exp.

␹2

OR

p Value

3rd Round

— 0.048

2nd Round DR*03 removed 0.007 NS

3rd Round

3rd Round

— 0.035

Abbreviations: Exp. ⫽ expected; HLA ⫽ human leukocyte antigen; MG ⫽ myasthenia gravis; ND ⫽ not determined; NS ⫽ not significant; Obs. ⫽ observed; OR ⫽ odds ratio.

Concerning HLA class II specificities, the increased frequency of DRB1*09 (OR ⫽ 5.9) observed in the total group of Venezuelan MG patients, has been reported elevated in Japanese patients with infantile MG [9], in adult Chinese patients [10, 23], and more recently also in Caucasoid patients [21]. Because HLA-DR9 is absent in isolated Amerindians, this allele is probably of Caucasian or African-American origin in Venezuelans. No secondarily associated class II allele could be found in the overall series of MG patients applying the RPE test, nor special interactions between two factors using Svejgaard’s 2⫻4 tables [16]. Nevertheless, the analysis confirmed the significant linkage disequilibrium between DRB1*03 DQB1*02 and DQA1*05 in our patients and controls. HLA class I specificities in the total group of MG patients revealed increased frequencies of A*31, B*08, B*39, B*40, C*15, and C*17 in patients compared with control patients. Sequential removal of HLA-B alleles when RPEs were looked for indicated that B8 is the allele with the largest contribution in the overall MG patients followed by B39 and B40. The increased frequencies of some of these class I and class II antigens in the total

group of patients can be explained when patients are analyzed according to thymic pathology. Thus, our results confirm previous studies reporting that patients with thymic hyperplasia, all of them except one being females with early onset of the disease, have increased frequencies of B*08, DRB1*03, DQA1*05, and DQB1*0201; HLA variants reported in high frequency in Caucasian MG patients. Even though it was not expected due to the borderline statistical significance observed for most of the associated alleles, the tests to identify those with the strongest association to MG in our patients detected DRB1*13 and B*38 as possible predisposing secondarily associated alleles in patients with hyperplasia. Two-locus haplotypes observed in Venezuelan MG patients corroborate increased frequencies of individual specificities. Thus A*01 B*08 and B*08 DRB1*03 haplotypes are very probably part of the ancestral suprahaplotype A1 B8 DRB1*0301 DQA1*0501 associated with MG in European and North American Caucasian populations. Venezuelan patients with thymic hyperplasia have also revealed increased frequencies of HLA-A*31 and C*17, whereas those with thymoma are

HLA Polymorphisms in Venezuelan Patients With Myasthenia Gravis

probably responsible for the increased frequency of B*39. Decreases in DRB1*03 and DQA1*05 among this last group of MG patients has been reported previously [8, 24]. Our findings in a group of ethnically mixed MG patients reflect the heterogeneity of our population and of the patients tested, and demonstrate the existence of various well-known associations with different HLA genes already described in MG patients of Caucasian and/or Oriental ethnic background. These results, together with several others performed in different populations, emphasize the need to better understand the disease and its clinical manifestations and suggest either a limited influence of the HLA genes involved or that these might be only markers of non-HLA genes responsible for the susceptibility or resistance effect. Thus, the etiology of MG may involve different HLA specificities together with several other non-HLA gene families playing important roles in the development of this complex autoimmune disease. REFERENCES 1. Carlsson B, Wallin J, Pirskanen R, Matell G, Smith CIE: Different HLA DR-DQ associations in subgroups of idiopathic myasthenia gravis. Immunogenetics 31:285, 1990. 2. Calhoun RF, Ritter JH, Guthrie TJ, Pestronk A, Meyers BF, Patterson GA, Pohl MS, Cooper JD: Results of transcervical thymectomy for myasthenia gravis in 100 consecutive patients. Ann Surg 230:555, 1999. 3. Shah AK: Myasthenia gravis. Med J 2:1, 2001. 4. Campbell RD, Milner CM: MHC genes in autoimmunity. Curr Opin Immunol 5:887, 1993. 5. Zelano G, Lino MM, Evoli A, Settesoldi D, Batocchi AP, Torrente I, Tonali PA: Tumor necrosis factor ␤ gene polymorphisms in myasthenia gravis. Eur J Immunogenet 25:403, 1998. 6. Pirskanen R: Genetic associations between myasthenia gravis and the HL-A system. J Neurol Neurosurg Psychiatry 39:23, 1976. 7. Fritze D, Hermann C, Nacim F, Smith G, Zeller E, Walford RL: HL-A antigens in myasthenia gravis. Lancet 1:240, 1973. 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. J Neuroimmunol 47:115, 1993. 9. Matsuki K, Juji T, Tokunaga K, Takamizawa M, Maeda H, Soda M, Nomura Y, Segawa M: HLA antigens in Japanese patients with myasthenia gravis. J Clin Invest 86:392, 1990. 10. Hawkins BR, Wy CL, Ekk C, Ay H: Strong association of HLA Bw46 with juvenile onset myasthenia gravis in Hong Kong Chinese. J Neurol Neurosurg Psychiatry 47:555, 1984.

59

11. Osserman KE, Genkins G: Studies in myasthenia gravis: review of a twenty-year experience in over 1200 patients. Mt Sinai J Med 38:497, 1971. 12. Miller S, Dykes D, Polesky HA: A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 16:1215, 1988. 13. Bignon JD, Fernandez-Vin˜ a MA, Cheneau ML, Fauchet R, Schreuder GM, Clayton J, Marsh SGE, Charron D: HLA DNA class II typing by PCR-SSOP: 12th International Histocompatibility Workshop experience. In Charron D (ed): Genetic Diversity of HLA. Functional and Medical Implication. Volume 1. Paris: EDK, 1997. 14. Woolf B: On estimating the relation between blood group and disease. Ann Hum Genet 19:251, 1955. 15. Haldane JBS: The estimation and significance of the logarithm of a ratio of frequencies. Ann Hum Genet 20:309, 1956. 16. Svejgaard A, Ryder LP: HLA and disease associations. Detecting the strongest association. Tissue Antigens 43: 18, 1994. 17. Payami H, Joe S, Farid NR, Stensky V, Chan SH, Yeo PPB, Chea JS, Thomson G: Relative predispositional effects (RPEs) of marker alleles with disease: HLA-DR alleles and Graves disease. Am J Hum Genet 45:541, 1989. 18. Imanishi I, Akaza T, Kimura A, Tokunaga K, Gojobori T: Allele and haplotype frequencies for HLA and complement loci in various ethnic groups. In Tsuji K, Aizava M, Sasazuki T (eds): HLA 1991: Proceedings of the Eleventh International Histocompatibility Workshop and Conference. Volume 1. Oxford, UK: Oxford University Press, 1991. 19. Cao K, Hollenbach J, Shie X, Shi W, Chopek M, Ferna´ ndez-Vin˜ a A: Analysis of the frequencies of HLA-A, B and C alleles and haplotypes in the five major ethnic groups of the United States reveals high levels of diversity in these loci and contrasting distribution patterns in these populations. Hum Immunol 62:1009, 2001. 20. Navaneetham D, Penn AS, Howard JF, Conti-Fine BM: TCR-Vbeta usage in the thymus and blood of myasthenia gravis patients. J Autoimmunity 11:621, 1998. 21. Giraud M, Beaurain G, Yamamoto AM, Eymard B, Tranchant C, Gajdos P, Garchon HJ: Linkage of HLA to myasthenia gravis and genetic heterogeneity depending on anti-titin antibodies. Neurology 57:1555, 2001. 22. Makhatadze NJ, Franco MT, Layrisse Z: HLA class I and class II allele and haplotype distribution in the Venezuelan population. Hum Immunol 55:53, 1997. 23. Chan SH, Tan CB, Lin YN, Wee GB, Degli-Esposti MA, Dawkins RL: HLA and Singaporean Chinese myasthenia gravis. Int Arch Allergy Immunol 101:119, 1993. 24. Franciotta D, Cuccia M, Dondi E, Piccolo G, Cosi V: Polymorphic markers in MHC class II/III region: a study on Italian patients with myasthenia gravis. J Neurol Sci 190:11, 2001.