Estrogen receptor gene polymorphism in Japanese patients with multiple sclerosis

Journal of the Neurological Sciences 179 (2000) 70–75 www.elsevier.com / locate / jns

Estrogen receptor gene polymorphism in Japanese patients with multiple sclerosis a, a b a a Masaaki Niino *, Seiji Kikuchi , Toshiyuki Fukazawa , Ichiro Yabe , Kunio Tashiro a

Department of Neurology, Hokkaido University Graduate School of Medicine, Kita-15 Nishi-7, Kita-ku, Sapporo 060 -8638, Japan b Hokuyukai Neurology Hospital, Sapporo, Japan Received 14 January 2000; received in revised form 24 May 2000; accepted 25 July 2000

Abstract Estrogen has been reported to have immunosuppressive functions, and to inhibit the progression of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). Since estrogen shows its biological effects via estrogen receptors (ER), we investigate the possible role of ER genes (ERG) in the pathogenesis of MS. PvuII and XbaI polymorphisms in ERG were detected by PCR-RFLP from the DNA of 79 conventional MS patients and 73 healthy controls. The [P] allele in the profiles in PvuII was significantly more prevalent in MS patients than in the controls (P,0.0005). In the study of XbaI polymorphism, the onset age of MS patients with the Xx genotype was earlier than that of the xx genotype group (mean age6S.D.; 22.6068.04, and 27.4969.14, respectively) (P,0.05) by ANOVA followed by Fisher’s PLSD. Although the Xx genotype group tended to earlier onset age than the XX genotype group (29.60611.10), this difference did not reach. On the basis of these results, PvuII polymorphism might be associated with susceptibility to MS, and XbaI polymorphism with onset age of MS. ERG polymorphism should be further studied in other populations to improve strategies for treatment of MS.  2000 Published by Elsevier Science B.V. Keywords: Estrogen receptor gene; Multiple sclerosis; Polymorphism; Susceptibility gene; Japanese

1. Introduction In autoimmune diseases such as multiple sclerosis (MS) and rheumatoid arthritis (RA), a distinct characteristic is the preponderance of female patients of reproductive age [1,2]. During pregnancy, in particular during the last trimester, both RA and MS tend to be ameliorated but then are often exacerbated postpartum [3–5]. Although the reason for this is unknown, sex hormones, sex chromosomes, and behavior and / or sex-determined neuroendocrine factors are all likely to play a role. Recent studies have shown the presence of estrogen receptors on cells involved in the immune response [6]. Furthermore, estradiol pre*Corresponding author. Tel.: 181-11-700-5375, ext. 6028; fax: 18111-700-5356. E-mail address: [email protected] (M. Niino).

vented injury-induced downregulation of bcl-2 expression in neural tissues [7]. In in vivo studies, long-term treatment with high levels of 17b-estradiol led to a dramatically delayed onset of experimental autoimmune encephalomyelitis (EAE), and mice treated with estriol experienced significantly decreased severity of EAE and delayed onset of disease [6,8]. The biological actions of estrogen receptors (ER) appear to be mediated through internuclear hormone-receptors [9], and recently the structure of the estrogen receptor genes (ERG)-a,b have been determined [10,11]. An association between bone mineral density (BMD) and ERG-a polymorphism has been reported [12,13]. In PvuII and XbaI polymorphism of ERGa, an association with breast cancer, Parkinson’s disease and Alzheimer’s disease was reported [14,15]. In BMD, an association with vitamin D receptor gene (VDRG) polymorphism, one of the steroid hormones, has been sug-

0022-510X / 00 / $ – see front matter  2000 Published by Elsevier Science B.V. PII: S0022-510X( 00 )00381-6

M. Niino et al. / Journal of the Neurological Sciences 179 (2000) 70 – 75

gested, and we previously reported that VDRG polymorphism might increase susceptibility to MS [16]. The recent full genome screening studies for MS suggested that a multifactorial etiology, including both environmental and multiple genetic factors of moderate effect, was more likely than an etiology consisting of simple mendelian disease gene [17–19]. In the present study, we investigated the association between ERG polymorphism and MS, with consideration given to the disease’s clinical course and severity, magnetic resonance imaging (MRI) findings, oligoclonal bands (OCB) in the cerebrospinal fluid (CSF) and human leukocyte antigens (HLA).

2. Patients and methods

2.1. Patients and healthy individuals Selected for this study were 79 unrelated Japanese patients with relapsing and remitting type (male 14, female 32) or secondary progressive type (male 8, female 25) MS who, after having been observed for at least 1 year, were diagnosed as having clinically definite MS according to the criteria of Poser et al. [20] (Table 1). They were ‘conventional’ MS patients as previously described [21–23], quite similar to those of Western MS patients. The mean6S.D. of their EDSS was 3.462.6. The control group was composed of 16 unrelated healthy men and 57 women

Table 1 Clinical profiles of MS patients Total (n579) Male:female Age a Age at onset a Course b Duration a EDSS c MRI d

OCB e positivity a

R-R S-P

I II III IVa IVb V

1:2.6 36.5611.5 25.569.1 46 (58.2%) 33 (41.8%) 11.168.9 3.562.7 0 (0.0%) 0 (0.0%) 10 (12.7%) 14 (17.7%) 7 (8.9%) 48 (60.8%) 27 / 48 (56.3%)

Mean (years)6S.D. R-R, relapsing-remitting course; S-P, secondary progressive course. c EDSS, expanded disability status scale of Kurtzke; mean EDSS6S.D. d Grading of MRI: grade I, no cerebral lesions in the white matter; grade II, only subcortical lesions without periventricular lesions; grade III, one to several periventricular lesions of small or moderate size; grade IVa, grade III plus subcortical lesions, with a total number less than 10; grade IVb, grade IVa plus one or two large lesions; grade V, widely distributed subcortical or periventricular white matter lesions, with a total of 10 or more lesions consisting of more than two large confluent lesions. e OCB, oligoclonal band in the cerebrospinal fluid. b

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ranging from 20 to 58 years of age (mean6S.D., 34.569.8). All patients and controls were Japanese and were residents of Hokkaido, the northernmost island of Japan [24]. The differences in sex ratio and age between the patients and controls were not significant (P50.3989 and P50.2553, respectively).

2.2. Analysis of ERG polymorphism After informed consent was obtained from each subject, high-molecular weight DNA was extracted from their peripheral blood cells. Polymerase chain reaction (PCR) amplification was performed on the region containing the PvuII and XbaI polymorphisms. The two polymorphisms were located at the same intron 1 of the ERG, and the same primers were used. The polymorphism was detected using two primers (forward, 59-CTGCCACCCTATCTGTATCTTTTCCTATTCTCC-39; reverse, 59-TCTTTCTCTGCCACCCTGGCGTCGTTATCTGA-39). After PCR-amplification of genomic DNA using a PJ9600 thermal cycler (Perkin-Elmer), the PCR products were digested by the respective restriction enzyme (PvuII or XbaI, Takara, Tokyo, Japan). Polymorphism was analyzed using a Mupid  -3 electrophoresis system (Cosmo Bio.). According to Mizunuma et al. [13] and Isoe-Wada et al. [14], either of these two enzymes detects a dimorphism of ERG. PCR was carried out in a total volume of 15 ml, containing 60 ng of genomic DNA, 5 pmol of each primer, 250 mM dNTP, 10 mM KCl, 20 mM Tris–HCl, pH 8.2, 1.5 mM MgCl 2 , 1.7 M N,N,N-trimethylglycine, and 0.12 U Taq polymerase (AmpliTaq; Perkin-Elmer). The PCR reaction mixtures were then denatured at 958C for 5 min., followed by 30 cycles of denaturation at 958C for 1 min, annealing at 618C for 1 min, extension at 728C for 1 min, and final elongation for 10 min using a PJ9600 thermal cycler (Perkin-Elmer). Each PCR product was thereafter digested by restriction enzyme PvuII or XbaI at 378C for 1 h. Eight microliters of each PCR product was mixed with 1 ml loading buffer (25% ficoll, 0.25% bromophenol, 0.25% xylene cyanol FF, containing 1 mM of EDTA) and electrophoresed for 40 min through a 2% agarose gel at 100 V in 13 TAE buffer containing 5 ml of ethidium bromide (10 mg / ml). Each polymorphism was determined using a Thermal Imaging System FTI-500  (Fuji Film).

2.3. Genomic HLA typing and MRI A nonisotopic oligotyping method using reverse dot blot hybridization was performed for HLA class II DRB1 and DPB1 alleles (INNO-LiPA HLA kit; Imnogenetics). This method discriminates between the 69 DRB1 and 47 DPB1 alleles. In the present study, DRB1 alleles were determined in 71 MS patients and 67 controls, and DPB1 alleles in 61 patients and 67 controls [16].

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2.4. Magnetic resonance imaging ( MRI) MRI analysis were performed in all 79 patients, and all scans were evaluated by the same neuroradiologist (KM) without knowledge of the clinical profiles [22].

2.5. Oligoclonal IgG band ( OCB) analysis OCB analyses were performed in 48 patients. CSF and serum samples were coded and sent for blind analysis to the laboratory at the Division of Clinical Chemistry of Vancouver Hospital and Health Sciences Center in Canada. An experienced observer, who had detected OCB in numerous patients, tested all of the samples for OCB by a previously described method [25]. The samples were tested by isoelectric focusing and silver staining visualization with a Resolve CSF kit (Isolab, USA).

Table 2 ERG polymorphism in MS patients and controls MS patients (n579)

Controls (n573)

Genotype frequencies (PvuII)* PP 15 (19.0%) Pp 49 (62.0%) pp 15 (19.0%)

6 (8.2%) 32 (43.8%) 35 (47.9%)

Allele frequencies (PvuII)** P 79 (50%) p 79 (50%)

44 (30.1%) 102 (69.9%)

Genotype frequencies (XbaI) XX 5 (6.3%) Xx 35 (44.3%) xx 39 (49.4%)

7 (9.6%) 32 (43.8%) 34 (46.6%)

Allele frequencies (XbaI) X 45 (28.5%) x 113 (71.5%)

46 (31.5%) 100 (68.5%)

*P,0.001; **P,0.0005; odds ratio, 2.32; 95% confidence interval, 2.03–2.65.

2.6. Statistical analysis Comparisons between the various alleles of patients with MS and those of the controls were made using the x 2 -test for two-by-two or two-by-three tables and Fisher’s exact test. The Student’s t-test, the x 2 -test and Fisher’s exact test were used to compare the clinical characteristics and MRI findings in the MS subgroups. Statistical analysis between genotype ERG polymorphism and onset age of MS was tested by analysis of variance (ANOVA) followed by Fisher’s protected least significant difference (PLSD).

3. Results

3.1. PvuII and XbaI genotype and allele frequencies The proportions of the three PvuII genotypes (PP, Pp, pp) and XbaI genotypes (XX, Xx, xx) were tabulated (Table 2). In control subjects, genotype frequencies conformed to Hardy–Weinberg expectations. We found that the rate of PP and Pp genotype was significantly higher in MS patients than in the controls (P,0.001). The [P] allele was more frequently represented in the MS patient group (50.0%) compared with the control group (30.1%) (P, 0.0005; odds ratio, 2.32; 95% confidence interval (CI), 2.03–2.65); however, the prevalence of XbaI genotype and allele frequencies did not show any differences between MS patients and controls.

3.2. PvuII polymorphism and MS Among the 79 patients, there was no association between PvuII polymorphism and clinical background, including such factors as age at blood sampling, age at onset,

disease duration, MRI findings, and EDSS (data not shown). Clinical course and disability, considered together with the duration of disease from onset to blood sampling calculated by a previously applied method [25], were also not associated with the polymorphism (data not shown).

3.3. XbaI polymorphism and onset age of MS In the relation between XbaI polymorphism and age at onset of MS, the onset ages of the patients with XX, Xx, and xx genotypes were (mean age6S.D.) 29.60611.10, 22.6068.04, and 27.4969.14, respectively (Table 3). The Xx genotype patients showed earlier onset than the xx genotype groups by ANOVA followed by Fisher’s PLSD (P,0.05). Xx genotype patients tended to show earlier onset than the XX genotype groups (P50.0996), but this was not significant and may have been due to the small number of XX genotype patients (n55).

3.4. PvuII and XbaI haplotype and MS No significant difference in the proportion of PvuII and XbaI haplotypes between MS patients and controls was Table 3 Age of disease onset in XbaI polymorphism Genotype

No. of patients

Age at onset (mean age6S.D.)

XX Xx xx

5 35 39

29.60611.10 22.6068.04* 27.4969.14

*P,0.05 versus xx. Statistical analysis was tested by ANOVA followed by Fisher’s PLSD.

M. Niino et al. / Journal of the Neurological Sciences 179 (2000) 70 – 75

detected. The study of clinical background also revealed no association with haplotype, including such factors as disease onset age (data not shown).

4. Discussion Although MS improves clinically during pregnancy, the mechanisms underlying this improvement are unknown [3,4]. In a study of estrogen, a hormone associated with pregnancy, estriol was found to increase IL-10 production [8]. Estrogen itself was found to activate the differentiation of extrathymic NK-T cells in the liver [26]. NK-T cells produce large amounts of IL-4 [27] and thereby direct an immune response. Taken together, these findings suggested that estrogen might demonstrate a shift toward humoral (Th2) immunity. In a recent study, 17a-ethinylestradiol partially suppressed the clinical signs and symptoms of EAE and caused a mild suppression of the number of infiltrates in the central nervous system without regulation of the immune system via a glucocorticoid side effect [28]. In the ER, the intranuclear receptor through which estrogen mediates, two subtypes (a, b) have been reported [10,11]. Furthermore, some polymorphisms in ERG-a have been identified, and several reports have appeared concerning the relation between some ERG-a polymorphisms and BMD, breast cancer, Alzheimer’s disease and Parkinson’s disease. In the study on the relation between PvuII polymorphism and breast cancer, breast cancer patients who were homozygous for the [P] allele were more likely to have ER-negative tumors [15]. In the neurodegenerative diseases, the frequency of the [P] allele in the Parkinson’s disease with dementia group as well as in the Alzheimer’s disease group was higher than that in control subjects [14]. On the other hand, several studies have investigated the association between ERG-a polymorphism and BMD [12,13,29]. Kobayashi et al. showed that PP or Pp genotypes had a tendency to have lower bone mineral density than pp genotype [12]. This data suggested the possibility that the PP or Pp genotypes might diminish the effect of estrogen. These results, together with our data, suggests that the susceptibility of the PP or Pp genotype to MS might be determined by a diminution of the immunomodulatory effect by estrogen. In a study of the relation between XbaI polymorphism and BMD with premenopausal women, the Xx genotype had significantly higher BMD than the other genotypes, whereas in the late-stage pregnancy group, no difference in BMD was shown in XbaI polymorphism. The Xx genotype had a tendency toward decreased BMD with aging. From these data, Mizunuma et al. suggested that the Xx genotype in ERG was more dependent on estrogen levels and might be more affected by slight changes in estrogen levels than the other genotypes [13]. MS patients with the Xx genotype had a younger onset of disease than those of the

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xx genotype, and tended to have a younger onset than those with XX genotypes. Taken together, the XbaI genotype may affect the age of disease onset, and the Xx genotype may possibly be associated with the earlier onset of MS. However, it has remained unclear why the heterozygote genotype would have altered expression. This question is beyond the present study, and further study is needed. Although to date four groups have completed systemic searches for linkage in MS, the chromosome 6q25.1, on which ERG-a occurs, has not been reported to reach statistical significance in transmission disequilibrium studies [17–19,30]. However, some studies have clearly indicated that there is no single major locus determining susceptibility to MS [17–19]. Furthermore, population association studies that compare the frequency of variants between cases and controls may be more effective and have greater power than transmission disequilibrium studies for common genetic variants having weak effect [31]. ERG may possibly be associated with MS. The ERG is organized into eight exons and seven introns extending over approximately 140 kilobases [32]. The restriction sites of the two enzymes are located at the same intron 1 of the ERG. Recent studies have revealed that some introns contain regulatory sequences such as enhancers [33,34], and more recently it has been shown that a polymorphism within the first intron has a significant effect on the level of protein synthesis [34]. Nevertheless, it is still unclear how the intronic polymorphism affects ER function [35,36]. However, the present study concerning the association between the two polymorphisms in ERG and MS suggests the possibility that these polymorphisms might affect the pathogenesis of MS. Patients in this study had ‘conventional’ type MS, quite similar to that of Western MS patients. In a HLA study with MS, DRB1*1501 allele was associated with ‘conventional’ type MS in Japanese as well as in Western patients [37]. However, the association between MS and CTLA-4 polymorphism showed some differences [38,39], and it was reported that the frequency of cerebellar MRI lesions in Japanese patients was lower than that in white patients with MS [40]. Taken together, these results suggest that even in the same ‘conventional’ type MS, differences may exist between the different ethnic groups. Population association studies such as the present study can be confounded by genetic differences between ethnic groups (‘population stratification’) that can lead to spurious associations [31]. The role of ERG polymorphism should be further studied in other populations to obtain more adequate strategies for treatment of MS.

Acknowledgements We thank Professor K. Miyasaka, Department of Radiology of Hokkaido University Graduate School of Medi-

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cine. We also thank Professors S.A. Hashimoto and D. Secombe, and Mr. A. Jamani (research technologist), Vancouver Hospital and Health Science Center, for the oligoclonal band testing. We thank Mr. T. Sasaki (research technologist), Sapporo City Hospital, for the HLA typing. We also thank Dr. T. Yanagawa of Nerima General Hospital, Tokyo, for his excellent help. This work was supported, in part, by a research grant from the Research on Brain Science, the Ministry of Health and Welfare of Japan.

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