Factors Influencing the Clinical Type and Course of Myasthenia Gravis Masatoshi Hayashi, MD, Kaichi Kida, MD, Shunro Sonoda, MD Hiroo Inoue, MD and Hiroshi Matsuda, MD
Myasthenia gravis is considered to be an autoimmune disease in which several factors reciprocally influence the clinical type and course. We investigated the relative importance of the following factors: anti-acetylcholine receptor antibody (A ChR A b), HLA, age at onset, autoimmunity, thymic abnormality, duration of treatment, change in AChR Ab titer and immunosuppressive therapy. The pretreatment-AChR Ab titer and HLA were shown to significantly influence the clinical type. On the other hand, the age at onset significantly influenced the clinical course. The finding that with an onset at less than 5-year-old there was a tendency for a good prognosis suggests an association between the immaturity of the muscle and immune systems, and the clinical course. Key words: Acetylcholine receptor antibody (AChR Ab), age at onset, HLA, myasthenia gravis (MG) and thymus. Hayashi M, Kida K, Sonoda S, Inoue H, Matsuda H. Factors influencing the clinical type and course of myasthenia gravis. Brain Dev 1992; 14:88-93
It is well known that the anti-acetylcholine receptor antibody (AChR Ab) plays an important role in the pathogenesis of myasthenia gravis (MG), and that measurement of the AChR Ab titer is useful for both the diagnosis and evaluation of the clinical course of MG [1-3]. However, recent studies have led to the speculation that some factor other than AChR Ab may be involved in the pathogenesis of MG, because 10% of MG patients have no detectable AChR Ab and because the clinical course in some patients appears to be independent of the AChR Ab titer [1-6]. HLA and the thymus might be involved in the pathogenesis of MG. Compston et al found in a population study on Caucasians that MG patients can be separated into two groups; younger females and older males [7] . The younger female group was shown to have high incidences of thymic hyperplasia, and HLA-AI, B8 and DR3, while the older male group was shown to have high incidences of
From the Department of Pediatrics, Ehime University School of Medicine, Ehime (MH, KK, HM); Department of Virology, Faculty of Medicine, Kagoshima University, Kagoshima (SS); and Ehime Prefectural Institute for Public Health, Ehime (HI). Received for publication: October 9,1991. Accepted for publication: February 4,1992. Correspondence address: Dr. Masatoshi Hayashi, Department of Pediatrics, Ehime University School of Medicine, Shigenobu-cho, Onsen-gun, Ehime 791-02, Japan.
thymomas, and HLA-A3, B7 and DR2. Otherwise MG is also well known to exhibit heterogeneity in other criteria, that is, ocular and generalized MG. Ocular and generalized MG are considered to have distinct pathogenetic mechanisms [8, 9], based on the existence of a sex difference, Le., a male preponderance in ocular MG, a different response to drugs, namely, ocular MG shows a good response to steroid hormones but a relatively poor response to anticholinesterase agents, and the detection of a thymic abnormality mainly in generalized MG. In order to explain the pathogenetic difference between ocular and generalized MG, the titer and characteristics of AChR Ab was investigated in several groups. The AChR Ab titer was reported to be high in generalized MG but low in ocular MG [1, 3-5], while some groups [8, 10] reported that there might be a qualitative difference between ocular and generalized MG as well as a quantitative difference. Our previous kinetic study involving bovine ocular and foot muscle created a little stir as to the pathogenesis for MG by showing that AChR Ab in ocular MG patients had a significantly higher affinity for foot muscle than for extraocular muscle, which indicates that the difference between ocular and generalized MG cannot be explained by the binding characteristics of AChR Ab as to muscle antigens [11]. Furthermore, we studied HLA in MG patients in order to address the question of the distinct genetic backgrounds of the two MG types. Ocular MG was shown to have a high incidence of HLA-DRw9 and a low
incidence of autoantibodies, whereas generalized MG was shown to have a high incidence of both HLA-DRw8 and autoantibodies [12] . On the other hand, MG patients exhibit different clinical courses, which are independent of the clinical type; some patients go into remission after treatment with anticholinesterase agents, while others attain remission following immunosuppressive therapy such as thymectomy and steroid therapy. We have also reported that the immunogenetic background might reflect the clinical course as well as the clinical type [13] . In this study, we studied the influence of various factors on the clinical type and course, such as the age at onset, autoimmunity, thymic abnormality, duration of treatment, change in the AChR Ab titer and immunotherapy, besides the pretreatment-AChR Ab titer and HLA. These factors might interact. We tried to elucidate the key factors by not only univariate but also multivariate analysis, using a logistic regression method. SUBJECTS Fifty-eight Japanese MG patients (24 men and 34 women), ranging in age at onset from 4 months to 72 years (mean: 24.3 years), participated in this study after informed consent was obtained. The diagnosis of MG was based on the clinical features, results of edrophonium testing and evoked e1ectromyographic findings [14]. MG was classified into two groups: the ocular type, in which the symptoms were restricted to the ocular muscles, and the generalized type, in which the symptoms were manifested in skeletal, bulbar and/or respiratory muscles. The ocular MG patients were followed for 2 years or more. A thymic abnormality was identified on the basis of plain chest X-rays and CT scans, in accordance with the method of Baron et al [15]. Of the 16 MG patients with an abnormal thymus, 12 underwent thymectomy and were proven to have a histological abnormality: 5 hyperplasias and 7 thymomas. The patients were first treated with anticholinesterase agents for 4-5 months and then immunosuppressive therapy (either steroid or thymectomy or both) if the clinical symptoms were not improved by the anticholinesterase agents or if the patients had trouble in their daily life. For estimation of the clinical effect, we classified the MG patients into 4 groups based on the classification system of Millichap and Dodge [16]; A: complete remission on no medication, B: remission on drugs, C: improvement but no remission, and D: condition the same or worse. We assessed the clinical effect by comparing the clinical symptoms and AChR Ab titer before and after treatment, the mean duration of which was 3.8 ± 2.8 years, with a range of 1-14 years, except for one case of 28 years. Twenty-seven patients with MG were treated with anticholinesterase agents only, while 31 patients also received immunosuppressive therapies. Eighty-five percent
(23/27) of the patients treated with anticholinesterase agents only attained remission, while 65% (20/31) of those patients treated with immunosuppressive therapies attained remission, which is defined as A + B in the classification system of Millichap and Dodge [16]. Two children and 7 adult patients with MG were excluded from the clinical course study, because they had no detectable AChR Ab before treatment or no blood sample was collected after the treatment. METHODS HLA type and autoantibodies We investigated the HLA haplotypes, AChR Ab titer and autoantibodies in 58 MG patients before beginning the treatment. We also measured the AChR Ab titer after the treatment in 49 of those MG patients and calculated the before: after ratio of the AChR Ab titers. HLA typing for the A, B, C and DR loci was performed by the standard NIH microlymphocytotoxicity technique using the typing sera of the 8th International Histocompatibility Workshop or local sera standardized against these sera, as described previously [13]. The controls were 150 healthy unrelated Japanese volunteers. The AChR Ab titer was measured by the method of Appel et al [17] using denervated rat muscle AChR as the antigen after partial modification [13]. Four kinds of autoantibodies were measured: antinuclear antibody and antistriated muscle antibody were assayed by an indirect immunofluorescence technique, while antithyroglobulin antibody and antithyroid microsome antibody were measured by a passive hemagglutination method. Autoimmunity was defined as "positive" if one and more of those four autoantibodies were detected, and "negative" if none of them was detected. Statistics The factors influencing the clinical type in the 58 MG patients were studied by means of univariate statistic analysis and multivariate logistic regression analysis. Simple associations among factors were studied by means of the chi-square test, analysis of variance or Student's t-test. We used LOG 1ST of SAS for multivariate analysis [18]. The explanatory variables for the clinical type study were the age at onset, sex, AChR Ab titer before treatment, autoimmunity, HLA-DR type and thymic abnormality. HLA-DR type was assessed for the combination of HLA-DRw8 and 9 based on the previous report [12]. They were classified into some levels, for which we used "dummy" variables. The age at onset was classified into the three levels, 0-5, 6-15 and over 16 years old, autoimmunity into two categories, "positive" and "negative," HLA-DR into four categories, DRw8/DRw9, DRw8/not DRw9, DRw9/not DRw8 and not DRw8/not DRw9, and thymic abnormality into two categories,
Hayashi et al: Factors influencing myasthenia 89
Table 2 Logistic regression analysis of the clinical type
Table 1 Factors influencing the clinical type (AJ Factoll
TypeofMG Ocular Generalized
Age at onset Child Adult
20b 10
10 18
Sex Male Female
13 17 11
Autoimmunity (+) (-)
HLA-DR DRw8/w9 DRw8/not9 DRw9/not8 DRwnot8/not9 Thymic abnormality (+) (-)
(BJ
Factoll
AssessmentC x2
Variable
-0.874 1.946
1.221 2.134
2.50 (df = 2)
1.283
1.090
1.39 (df = 1)
AChR Ab titer (before)
- (not converge)
- (not converge)
25.82 (df = 1)**
Autoimmunity
0.424
1.334
0.10 (df = 1)
-0.351 -2.210 1.795
1. 758 1.544 1.766
8.86 (df = 3)*
-1.692
1.473
1.32 (df = 1)
Age of onset -5 6-15
11 17
0.098
Sex
19
18 10
4.42 (p < 0.05)
5 5 14 6
6 15 5 2
11.299 (p < 0.05)
HLA-DR DRw8/w9 DRw8/not9 DRw9/not8
2 28
14 14
11.531 (p < 0.01)
Thymic abnormality
Age at onset (years old)
17.2
±
20.6 d
31.9
±
23..9
AChR Ab titer before treatment (nM)
0.24
±
0.24
2.98
±
3.67
Assessmente
(p
The predicted values for each variable were statistically assessed based on the fact that they follow a chi-square distribution within the appropriate degree of freedom. The x' value was significantly high for the HLA-DR type *(p < 0.05) and the AChR Ab titer **(p < 0.01) in spite of no convergence.
< 0.01)
Factor a: see Methods for details, Figures b : indicate the numbers of patients statistically assessed by means of the chi-square test C in Table lA, Years old (mean ± SD)a for the age at onset and nM for the AChR Ab titer were statistically assessed by means of Student's t-test e in Table lB.
present and absent. The age at onset was classified into two groups for univariate analysis: less than 15 years old as child and 16 years or older as adult. We also performed logistic regression analysis to study the factors influencing the clinical courses of the 49 MG patients for whom pre- and posttreatment AChR Ab titer data were available. The explanatory variables for the clinical course study were the clinical type, age at onset, sex, autoimmunity, HLA-DR type, thymic abnormality, AChR Ab titer before treatment, posttreatment/pretreatment ratio of the AChR Ab titer, duration of treatment and immunosuppressive therapy. Similarly, the clinical type was classified into two categories, ocular and generalized MG, and immunosuppressive therapy into two categories, "yes" and "no," for steroid therapy and thymectomy.
RESULTS As shown in Table 1, simple association analyses using the chi-square test showed significant correlations between
90 Brain & Development, Vol 14, No 2,1992
x2
1.666
5.56
TypeofMG Ocular Generalized
SE (Si)
2.710
Constant
< 0.05)
(p
Predicted ~i
the clinical type and the following factors: the age at onset (X2 = 5.56, p < 0.05), 67% of the juvenile patients having ocular MG and 65% of the adult patients having generalized MG; the autoimmunity status (X2 = 4.42, p < 0.05), 62% of the patients with autoantibodies having generalized MG and 66% of the patients without autoantibodies having ocular MG; the HLA-DR type (X 2 = 11.30, P < 0.05), HLA-DRw9 was frequent in ocular MG and HLA-DRw8 frequent in generalized MG; thymic abnormality (X2 = 11.53, p < 0.01), 88% of the patients with a thymic abnormality having generalized MG and 67% of the patients without any thymic abnormality having ocular MG. The unpaired Student's t-test showed a significantly low AChR Ab titer before treatment (p < 0.01) (Table 1). When logistic regression analysis of the clinical type was performed for the various explanatory variables, significance was found for the variables of the AChR Ab titer before treatment (X2 = 25.82, p < 0.01) and HLA-DR (X2 = 8.86, p < 0.05) (Table 2). We used the chi-square test to study the association of the clinical course with the following factors: the age at onset, clinical type, sex, autoimmunity, HLA-DR type, thymic abnormality and kind of immunosuppressive therapy. Only two factors, i.e., the age at onset (X2 = 10.01, p
Myasthenia gravis is considered to be an autoimmune disease in which several factors reciprocally influence the clinical type and course. We investigated the relative importance of the following factors: anti-acetylcholine receptor antibody (A ChR A b), HLA, age at onset, autoimmunity, thymic abnormality, duration of treatment, change in AChR Ab titer and immunosuppressive therapy. The pretreatment-AChR Ab titer and HLA were shown to significantly influence the clinical type. On the other hand, the age at onset significantly influenced the clinical course. The finding that with an onset at less than 5-year-old there was a tendency for a good prognosis suggests an association between the immaturity of the muscle and immune systems, and the clinical course. Key words: Acetylcholine receptor antibody (AChR Ab), age at onset, HLA, myasthenia gravis (MG) and thymus. Hayashi M, Kida K, Sonoda S, Inoue H, Matsuda H. Factors influencing the clinical type and course of myasthenia gravis. Brain Dev 1992; 14:88-93
It is well known that the anti-acetylcholine receptor antibody (AChR Ab) plays an important role in the pathogenesis of myasthenia gravis (MG), and that measurement of the AChR Ab titer is useful for both the diagnosis and evaluation of the clinical course of MG [1-3]. However, recent studies have led to the speculation that some factor other than AChR Ab may be involved in the pathogenesis of MG, because 10% of MG patients have no detectable AChR Ab and because the clinical course in some patients appears to be independent of the AChR Ab titer [1-6]. HLA and the thymus might be involved in the pathogenesis of MG. Compston et al found in a population study on Caucasians that MG patients can be separated into two groups; younger females and older males [7] . The younger female group was shown to have high incidences of thymic hyperplasia, and HLA-AI, B8 and DR3, while the older male group was shown to have high incidences of
From the Department of Pediatrics, Ehime University School of Medicine, Ehime (MH, KK, HM); Department of Virology, Faculty of Medicine, Kagoshima University, Kagoshima (SS); and Ehime Prefectural Institute for Public Health, Ehime (HI). Received for publication: October 9,1991. Accepted for publication: February 4,1992. Correspondence address: Dr. Masatoshi Hayashi, Department of Pediatrics, Ehime University School of Medicine, Shigenobu-cho, Onsen-gun, Ehime 791-02, Japan.
thymomas, and HLA-A3, B7 and DR2. Otherwise MG is also well known to exhibit heterogeneity in other criteria, that is, ocular and generalized MG. Ocular and generalized MG are considered to have distinct pathogenetic mechanisms [8, 9], based on the existence of a sex difference, Le., a male preponderance in ocular MG, a different response to drugs, namely, ocular MG shows a good response to steroid hormones but a relatively poor response to anticholinesterase agents, and the detection of a thymic abnormality mainly in generalized MG. In order to explain the pathogenetic difference between ocular and generalized MG, the titer and characteristics of AChR Ab was investigated in several groups. The AChR Ab titer was reported to be high in generalized MG but low in ocular MG [1, 3-5], while some groups [8, 10] reported that there might be a qualitative difference between ocular and generalized MG as well as a quantitative difference. Our previous kinetic study involving bovine ocular and foot muscle created a little stir as to the pathogenesis for MG by showing that AChR Ab in ocular MG patients had a significantly higher affinity for foot muscle than for extraocular muscle, which indicates that the difference between ocular and generalized MG cannot be explained by the binding characteristics of AChR Ab as to muscle antigens [11]. Furthermore, we studied HLA in MG patients in order to address the question of the distinct genetic backgrounds of the two MG types. Ocular MG was shown to have a high incidence of HLA-DRw9 and a low
incidence of autoantibodies, whereas generalized MG was shown to have a high incidence of both HLA-DRw8 and autoantibodies [12] . On the other hand, MG patients exhibit different clinical courses, which are independent of the clinical type; some patients go into remission after treatment with anticholinesterase agents, while others attain remission following immunosuppressive therapy such as thymectomy and steroid therapy. We have also reported that the immunogenetic background might reflect the clinical course as well as the clinical type [13] . In this study, we studied the influence of various factors on the clinical type and course, such as the age at onset, autoimmunity, thymic abnormality, duration of treatment, change in the AChR Ab titer and immunotherapy, besides the pretreatment-AChR Ab titer and HLA. These factors might interact. We tried to elucidate the key factors by not only univariate but also multivariate analysis, using a logistic regression method. SUBJECTS Fifty-eight Japanese MG patients (24 men and 34 women), ranging in age at onset from 4 months to 72 years (mean: 24.3 years), participated in this study after informed consent was obtained. The diagnosis of MG was based on the clinical features, results of edrophonium testing and evoked e1ectromyographic findings [14]. MG was classified into two groups: the ocular type, in which the symptoms were restricted to the ocular muscles, and the generalized type, in which the symptoms were manifested in skeletal, bulbar and/or respiratory muscles. The ocular MG patients were followed for 2 years or more. A thymic abnormality was identified on the basis of plain chest X-rays and CT scans, in accordance with the method of Baron et al [15]. Of the 16 MG patients with an abnormal thymus, 12 underwent thymectomy and were proven to have a histological abnormality: 5 hyperplasias and 7 thymomas. The patients were first treated with anticholinesterase agents for 4-5 months and then immunosuppressive therapy (either steroid or thymectomy or both) if the clinical symptoms were not improved by the anticholinesterase agents or if the patients had trouble in their daily life. For estimation of the clinical effect, we classified the MG patients into 4 groups based on the classification system of Millichap and Dodge [16]; A: complete remission on no medication, B: remission on drugs, C: improvement but no remission, and D: condition the same or worse. We assessed the clinical effect by comparing the clinical symptoms and AChR Ab titer before and after treatment, the mean duration of which was 3.8 ± 2.8 years, with a range of 1-14 years, except for one case of 28 years. Twenty-seven patients with MG were treated with anticholinesterase agents only, while 31 patients also received immunosuppressive therapies. Eighty-five percent
(23/27) of the patients treated with anticholinesterase agents only attained remission, while 65% (20/31) of those patients treated with immunosuppressive therapies attained remission, which is defined as A + B in the classification system of Millichap and Dodge [16]. Two children and 7 adult patients with MG were excluded from the clinical course study, because they had no detectable AChR Ab before treatment or no blood sample was collected after the treatment. METHODS HLA type and autoantibodies We investigated the HLA haplotypes, AChR Ab titer and autoantibodies in 58 MG patients before beginning the treatment. We also measured the AChR Ab titer after the treatment in 49 of those MG patients and calculated the before: after ratio of the AChR Ab titers. HLA typing for the A, B, C and DR loci was performed by the standard NIH microlymphocytotoxicity technique using the typing sera of the 8th International Histocompatibility Workshop or local sera standardized against these sera, as described previously [13]. The controls were 150 healthy unrelated Japanese volunteers. The AChR Ab titer was measured by the method of Appel et al [17] using denervated rat muscle AChR as the antigen after partial modification [13]. Four kinds of autoantibodies were measured: antinuclear antibody and antistriated muscle antibody were assayed by an indirect immunofluorescence technique, while antithyroglobulin antibody and antithyroid microsome antibody were measured by a passive hemagglutination method. Autoimmunity was defined as "positive" if one and more of those four autoantibodies were detected, and "negative" if none of them was detected. Statistics The factors influencing the clinical type in the 58 MG patients were studied by means of univariate statistic analysis and multivariate logistic regression analysis. Simple associations among factors were studied by means of the chi-square test, analysis of variance or Student's t-test. We used LOG 1ST of SAS for multivariate analysis [18]. The explanatory variables for the clinical type study were the age at onset, sex, AChR Ab titer before treatment, autoimmunity, HLA-DR type and thymic abnormality. HLA-DR type was assessed for the combination of HLA-DRw8 and 9 based on the previous report [12]. They were classified into some levels, for which we used "dummy" variables. The age at onset was classified into the three levels, 0-5, 6-15 and over 16 years old, autoimmunity into two categories, "positive" and "negative," HLA-DR into four categories, DRw8/DRw9, DRw8/not DRw9, DRw9/not DRw8 and not DRw8/not DRw9, and thymic abnormality into two categories,
Hayashi et al: Factors influencing myasthenia 89
Table 2 Logistic regression analysis of the clinical type
Table 1 Factors influencing the clinical type (AJ Factoll
TypeofMG Ocular Generalized
Age at onset Child Adult
20b 10
10 18
Sex Male Female
13 17 11
Autoimmunity (+) (-)
HLA-DR DRw8/w9 DRw8/not9 DRw9/not8 DRwnot8/not9 Thymic abnormality (+) (-)
(BJ
Factoll
AssessmentC x2
Variable
-0.874 1.946
1.221 2.134
2.50 (df = 2)
1.283
1.090
1.39 (df = 1)
AChR Ab titer (before)
- (not converge)
- (not converge)
25.82 (df = 1)**
Autoimmunity
0.424
1.334
0.10 (df = 1)
-0.351 -2.210 1.795
1. 758 1.544 1.766
8.86 (df = 3)*
-1.692
1.473
1.32 (df = 1)
Age of onset -5 6-15
11 17
0.098
Sex
19
18 10
4.42 (p < 0.05)
5 5 14 6
6 15 5 2
11.299 (p < 0.05)
HLA-DR DRw8/w9 DRw8/not9 DRw9/not8
2 28
14 14
11.531 (p < 0.01)
Thymic abnormality
Age at onset (years old)
17.2
±
20.6 d
31.9
±
23..9
AChR Ab titer before treatment (nM)
0.24
±
0.24
2.98
±
3.67
Assessmente
(p
The predicted values for each variable were statistically assessed based on the fact that they follow a chi-square distribution within the appropriate degree of freedom. The x' value was significantly high for the HLA-DR type *(p < 0.05) and the AChR Ab titer **(p < 0.01) in spite of no convergence.
< 0.01)
Factor a: see Methods for details, Figures b : indicate the numbers of patients statistically assessed by means of the chi-square test C in Table lA, Years old (mean ± SD)a for the age at onset and nM for the AChR Ab titer were statistically assessed by means of Student's t-test e in Table lB.
present and absent. The age at onset was classified into two groups for univariate analysis: less than 15 years old as child and 16 years or older as adult. We also performed logistic regression analysis to study the factors influencing the clinical courses of the 49 MG patients for whom pre- and posttreatment AChR Ab titer data were available. The explanatory variables for the clinical course study were the clinical type, age at onset, sex, autoimmunity, HLA-DR type, thymic abnormality, AChR Ab titer before treatment, posttreatment/pretreatment ratio of the AChR Ab titer, duration of treatment and immunosuppressive therapy. Similarly, the clinical type was classified into two categories, ocular and generalized MG, and immunosuppressive therapy into two categories, "yes" and "no," for steroid therapy and thymectomy.
RESULTS As shown in Table 1, simple association analyses using the chi-square test showed significant correlations between
90 Brain & Development, Vol 14, No 2,1992
x2
1.666
5.56
TypeofMG Ocular Generalized
SE (Si)
2.710
Constant
< 0.05)
(p
Predicted ~i
the clinical type and the following factors: the age at onset (X2 = 5.56, p < 0.05), 67% of the juvenile patients having ocular MG and 65% of the adult patients having generalized MG; the autoimmunity status (X2 = 4.42, p < 0.05), 62% of the patients with autoantibodies having generalized MG and 66% of the patients without autoantibodies having ocular MG; the HLA-DR type (X 2 = 11.30, P < 0.05), HLA-DRw9 was frequent in ocular MG and HLA-DRw8 frequent in generalized MG; thymic abnormality (X2 = 11.53, p < 0.01), 88% of the patients with a thymic abnormality having generalized MG and 67% of the patients without any thymic abnormality having ocular MG. The unpaired Student's t-test showed a significantly low AChR Ab titer before treatment (p < 0.01) (Table 1). When logistic regression analysis of the clinical type was performed for the various explanatory variables, significance was found for the variables of the AChR Ab titer before treatment (X2 = 25.82, p < 0.01) and HLA-DR (X2 = 8.86, p < 0.05) (Table 2). We used the chi-square test to study the association of the clinical course with the following factors: the age at onset, clinical type, sex, autoimmunity, HLA-DR type, thymic abnormality and kind of immunosuppressive therapy. Only two factors, i.e., the age at onset (X2 = 10.01, p
