Estrogen receptor gene polymorphism in a Chinese population with multiple sclerosis＊☆

Qingli Sun, Ruping Xie, Yu Fu, Xiaogang Li, Dongsheng Fan

Department of Neurology, Peking University Third Hospital, Beijing 100191, China

lNTRODUCTlON

Multiple sclerosis (MS) is a multifocal demyelinating disease affecting the central nervous system. MS is more prevalent in women, particularly during pregnancy[1-2].

Long-term estrogen administration at high levels in animals significantly delayed the onset of experimental autoimmune encephalomyelitis, and reduced the severity of the disease[3-5]. Estriol has been shown to inhibit the nuclear transcription factor kappa B, accordingly regulate some immune-related genes[6]. As such, we speculate that estrogen may be associated with humoral immunity, and thus may participate in the pathogenesis of MS.

Estrogen in the human body exerts its biological effects through the estrogen receptor (ER), and ER expression and function affect the biological effects of estrogen in the human body. ER ligands exert neuroprotective effects on experimental autoimmune encephalomyelitis through ER[7-9]. In addition, estrogen reduces the levels of matrix metalloproteinase 9 through ER,thus exerting a curative effect on MS and experimental autoimmune encephalomyelitis[10]. Different ER genotypes determine the alterations of ER expression and function in individuals, thereby affecting the ultimate effect of estrogen[11].

There are two subtypes of ER: ERα and ERβ. The human ERα gene is located on 6q24-27. This gene in intron 1 contains two point mutations: PvuII and XbaI restriction sites. Intron 1 includes enhancer,promoter and other important sequences,and point mutations affect ER expression and function[12]. The PvuII and XbaI restriction polymorphism of ERα contribute to breast cancer, Parkinson's disease,Alzheimer's disease, systemic lupus erythematosus and osteoarthritis[13-16]. However, reports of the correlation between ER gene polymorphism and the MS are rare. The current study sought to explore the PvuII and XbaI restriction polymorphism of ERα in 74 Chinese patients with MS, using polymerase chain reaction-restriction fragment-length polymorphism analysis.

RESULTS

Quantitative analysis of the involved subjects

We included 74 cases of Chinese patients with MS and 95 healthy Chinese subjects in this experiment. No participants were excluded during the experiment, and all subjects’ data were included in the final analysis.

Baseline data analysis in MS patients and control subjects

The baseline data between MS patients and controls are shown in Table 1. There were no significant differences in gender and age between the two groups (P ＞ 0.05).

Table 1 Comparison of baseline data between MS group and control group

Gene polymorphism and Hardy-Weinberg equilibrium test in MS patients and control subjects

PvuII restriction polymorphism: three genotypes, namely PP (1 band at 1.3 kb), Pp (3 bands at 1.3 kb, 850 bp and 450 bp), and pp (2 bands at 850 bp and 450 bp; Figure 1).XbaI restriction polymorphism: three genotypes, namely XX (1 band at 1.3 kb), Xx (3 bands at 1.3 kb, 910 bp and 390 bp), and xx (2 bands at 910 bp and 390 bp; Figure 2).

Figure 1 PvuII restriction polymorphism electrophoresis 1: PP genotype; 2, 3, 6: pp genotype; 4, 5, 7: Pp genotype;M: Marker.

Figure 2 XbaI restriction polymorphism electrophoresis.1, 2, 4: xx genotype; 3: XX genotype; 6, 7: Xx genotype;M: Marker.

Distributions of ER genotypes in the MS patients and control subjects were in accordance with the Hardy-Weinberg equilibrium, indicating that the gene frequencies reached a genetic equilibrium and were representative of the population (Table 2).

Table 2 Hardy-Weinberg equilibrium analysis of estrogen receptor genotype allele frequencies

Association analysis of the genotype and allele frequencies with MS in MS patients and control subjects

The distributions of ER gene PP, Pp, pp genotype frequency revealed significant differences between MS patients and control subjects (P ＜ 0.05), but no difference was observed between men and women (P ＞ 0.05). P and p allele frequency revealed significant differences between MS patients and control subjects (P ＜ 0.05). No significant differences were found between men and women in MS patients or control subjects, or in men between the MS patient group and the control group(P ＞ 0.05). However, the results revealed significant differences between women in the MS group and women in the control group (P ＜ 0.05; Table 3).

Table 3 Frequency distribution of PvuII genotype and allele in MS and control groups [n (%)]

There were no significant differences in the distribution of ER gene XX, Xx, xx genotypes and X, x allele frequencies between MS group and control group (P ＞ 0.05;Table 4).

Table 4 Frequency distribution of XbaI genotype and allele in MS and control groups [n (%)]

Joint analysis of ER gene PvuII and XbaI polymorphisms revealed that PpXx was dominant in the MS group, followed by ppxx and Ppxx; ppxx was dominant in the control group, followed by PpXx and Ppxx. Ppxx accounted for 24.3% of the MS group and 12.8% of the control group. ppxx accounted for 25.7% of the MS group and 44.7% of the control group, with significant differences between the two groups (P ＜ 0.05; Table 5).

Table 5 Frequency distribution of PvuII and XbaI genotypes in MS and control groups (%)

The PpXX, ppXX and ppXx genotypes were not found in either group. In the MS group, patient's age, the number of attacks and expanded disability status scale scores were not associated with ER genotypes (PP, Pp, pp and XX, Xx, xx) or allele frequency (P, p and X, x) (P ＞ 0.05).

DlSCUSSlON

The present results revealed that there were significant differences in the frequency distribution of ER gene P and p alleles between the MS group and the control group, while the X and x alleles exhibited no such differences. This finding is consistent with results of Niino et al[17], who reported that PvuII restriction polymorphism was associated with the occurrence of MS, and XbaI polymorphism with the onset age of MS. In 2002, Kikuchi et al[18]further confirmed that the P allele in female patients interacted with HLA-DRB1 ＊ 1501 to increase the risk of MS. A study in Finland also proposed that PvuII restriction polymorphism was related to MS in female patients, reporting that women carrying the pp genotype and HLA-DR2 were highly susceptible to MS[19]. This finding indicates that the pp genotype and HLA-DR2 may promote the development of MS in female patients[19].

The current analysis revealed that the frequency distribution of the P and p alleles exhibited significant differences in women between MS patients and the control group, while no significant difference was found between men in the two groups. This finding indicates that the difference between groups occurred as a result of the P and p allele distribution in women. This evidence suggests that women carrying the P allele are more susceptible to MS compared with women carrying the p allele.

However, a study in an Italian population reported no correlation between ER gene PvuII and XbaI polymorphisms and the onset of MS[20]. Another study involving 110 MS patients in Australia also reported no significant differences of the PvuII allele frequencies between the patient group and the control group[21]. We propose that this discrepancy in results may have been caused by differences in the genetic backgrounds between the different ethnic groups studied.

Liu et al[22]reported that pp-type and xx-type ER genes were the most commonly observed in a Chinese Han population, while the PP-type and XX-type were the least common. The current experiment obtained similar results:the pp-type and xx-type were dominant in the control group, while the PP-type and XX-type were rarely seen.

ER gene polymorphism is thought to vary between ethnic groups[22]. The population distribution has been found to be close between China and Japan, but greatly varies between China, Sweden, and the USA[23-25]. Specifically,the frequency of the pp-type in the Chinese population was found to be significantly higher than that in populations from the USA and Sweden. The frequency of the Pp-type in the Swedish population was significantly higher than that in China[10,25]. ppXX was not detected in the above studies. In addition, ppXx and PpXX are also rare, indicating that the pX haploid chromosome is rarely observed, and requires further study. In the current study,Ppxx frequency distribution in MS patients was significantly higher than that in the control group. This alteration was predominantly observed in male patients; the Ppxx genotype was observed in 33.3% of male MS patients, but only 13.2% of the control group. Taken together, this evidence indicates that the Ppxx genotype is a potential risk factor for MS in males.

In different social groups, population stratification may occur as a result of confounding variables, racial differences, sex ratio and age structure. As such,Hardy-Weinberg equilibrium analysis is necessary to verify differences between MS patients and the control group.

In summary, ER gene polymorphism was found to be associated with the pathogenesis of MS in a Chinese Han population. However, the sample size of this study was small, and previous findings regarding ER gene polymorphisms associated with MS are controversial. As such, further research involving large-sample,multi-center studies is required for firm conclusions to be drawn.

SUBJECTS AND METHODS

Design

A case-control study using polymorphism analysis.

Time and setting

The experiments were conducted from September 1999 to July 2010 in Peking University Third Hospital, China.

Subjects

A total of 74 MS patients were recruited from Peking University Third Hospital, China, from September 1999 to July 2010. Subjects included 27 males and 47 females,aged 14-76 years, with a mean age of 41.92 ± 12.95 years. The disease duration ranged from 3 months to 27 years, with a mean duration of 4.61 ± 2.82 years.

Inclusion criteria

All patients were diagnosed in accordance with McDonald diagnostic criteria for MS[26]: (1) two or more than two attacks, with two or more lesions as objective clinical evidence; (2) two or more than two attacks, with one lesion as objective clinical evidence, and spatial evidence for multiple episodes; (3) one attack, with two or more than two lesions as objective clinical evidence, and temporal evidence for multiple episodes; (4) one attack,with one lesion as objective clinical evidence, and spatial and temporal evidence for multiple episodes.

Exclusion criteria

Subjects who refused to collect blood samples or perform genetic studies were excluded from this study. The neurological deficits of MS patients were detected using expanded disability status scale (EDSS)[27]. All patients underwent head and spinal cord MRI.

The control group (n = 95) included 39 males and 56 females, aged 17-74 years, with a mean age of 45.32 ±10.67 years. Control subjects were unrelated healthy examinees from Peking University Third Hospital, China.

All subjects were of Han Chinese ethnicity, from various provinces in China. Experimental protocols were in line with the Administrative Regulations on Medical Institutions, published by the State Council of the People’s Republic of China[28]. The experimental methods were described in details to subjects or their families, who gave full cooperation and provided written informed consent. The study was approved by the Institutional Ethics Committee.

Methods

DNA extraction

Anticoagulant whole blood 5 mL was sampled from the right elbow vein of each MS patient and control, and genomic DNA was extracted. DNA concentration was detected using a UV spectrophotometer (Shanghai Mapada Instruments Co., Ltd., Shanghai, China).

ER gene polymorphism analysis

Genomic DNA was amplified with PCR. The total reaction volume of 25 μL contained 0.1 μg genomic DNA, 2.5 μL 10 × PCR buffer, 0.3 mmol/L of each upstream and downstream primer (upstream primer: 5’-CTG CCA CCC TAT CTT TTC CTA TTC TCC-3’, downstream primer:5’-TCT TTC TCT GCC ACC CTG GCG TCG ATT ATC TGA-3’; synthesized by Shanghai Sangon Biological Engineering Co., China), 0.2 mmol/L dNTPs, 1 U Tag DNA polymerase, and 13.5 μL triple-distilled water. A PCR instrument (Sparta, New Jersey, USA) was utilized for amplification reactions.

PCR conditions: 95°C denaturation for 5 minutes; 35 cycles of 94°C denaturation for 30 seconds, 61°C annealing for 60 seconds, and 72°C extension for 90 seconds; 72°C extension for 7 minutes. PCR product fragments were 1.3 kb. The amplified products (10 μL)were respectively digested with 6 U PvuII enzyme and 14 U XbaI enzyme (Sino-American Biotechnology Company, China) at 37°C for 2 hours. Specimens were separated in 2% ethidium bromide-stained agarose gel electrophoresis. The gel was placed in a UV projection instrument (Shanghai Changxing Equipment Factory,China) to analyze the genotypes.

The experiment was repeated once, and the results were judged by two physicians. Experiments were repeated if the results were inconsistent.

Statistical analysis

Measurement data were expressed as mean ± SD, and count data were expressed as percentages. Data were analyzed with SPSS 15.0 statistical analysis software(SPSS, Chicago, IL, USA). Genotype and allele frequencies were calculated with a gene counting method.ER allele frequencies and Hardy-Weinberg equilibrium analysis between different groups was analyzed using a fourfold table chi-square test. ER genotype was determined using an unpaired R × C table for the chi-square test. Patient’s age, the number of attacks and EDSS score between genotypes and alleles were compared with analysis of variance. A level of P ＜ 0.05 was considered to indicate statistically significant differences.

Author contributions:Qingli Sun was responsible for the implementation of experiments, statistical analysis and writing the manuscript. Ruping Xie was responsible for the study concept and design. Yu Fu was in charge of funding. Xiaogang Li and Dongsheng Fan provided technical and information support,and instructed the study.

Conflicts of interest:None declared.

Funding:This study was financially sponsored by the National Natural Science Foundation of China, No. 81071148.

Supplementary information:Supplementary data associated with this article can be found in the online version, by visiting www.nrronline.org, and entering Vol. 6, No. 19, 2011 after selecting the “NRR Current Issue” button on the page.

[1]Confavreux C, Hutchinson M, Hours MM, et al. Rate of pregnancy-related relapse in multiple sclerosis. Pregnancy in Multiple Sclerosis Group. N Engl J Med. 1998;339(5):285-291.

[2]Stuart M, Bergstrom L. Pregnancy and multiple sclerosis. J Midwifery Womens Health. 2011;56(1):41-47.

[3]Jansson L, Olsson T, Holmdahl R. Estrogen induces a potent suppression of experimental autoimmune encephalomyelitis and collagen-induced arthritis in mice. J Neuroimmunnol. 1994;53:203-207.

[4]Kim S, Liva SM, Dalal MA, et al. Estriol ameliorates autoimmune demyelinating disease: implications for multiple sclerosis.Neurology. 1999;52(6):1230-1238.

[5]Nashold FE, Spach KM, Spanier JA, et al. Estrogen controls vitamin D3-mediated resistance to experimental autoimmune encephalomyelitis by controlling vitamin D3 metabolism and receptor expression. J Immunol. 2009;183(6):3672-3681.

[6]Zang YC, Halder JB, Hong J, et al. Regulatory effects of estriol on T cell migration and cytokine profile: inhibition of transcription factor NF-kappa B. J Neuroimmunol. 2002;124(1-2):106-114.

[7]Tiwari-Woodruff S, Voskuhl RR. Neuroprotective and anti-inflammatory effects of estrogen receptor ligand treatment in mice. J Neurol Sci. 2009;286(1-2):81-85.

[8]Morales LB, Loo KK, Liu HB, et al. Treatment with an estrogen receptor alpha ligand is neuroprotective in experimental autoimmune encephalomyelitis. J Neurosci. 2006;26(25):6823-6833.

[9]Bebo BF Jr, Dehghani B, Foster S, et al. Treatment with selective estrogen receptor modulators regulates myelin specific T-cells and suppresses experimental autoimmune encephalomyelitis.Glia. 2009;57(7):777-790.

[10]Gold SM, Sasidhar MV, Morales LB, et al. Estrogen treatment decreases matrix metalloproteinase (MMP)-9 in autoimmune demyelinating disease through estrogen receptor alpha (ERalpha).Lab Invest. 2009;89(10):1076-1083.

[11]Hill SM, Fuqua SA, Chamness GC, et al. Estrogen receptor expression in human breast cancer association with an estrogen receptor gene restriction fragment length polymorphism. Cancer Res. 1989;49(1):145-148.

[12]Shaw JA, UdokangK, Mosquera JM, et al. Oestrogen receptor and beta differ in normal Human breast and breast carcinomas. J Pathol. 2002;198(4):450-457.

[13]Ushiyama T, Ueyama H, Inoue K, et al. Estrogen receptor gene polymorphism and generalized osteoarthritis. Rhenmatol. 1998;25(1):134-137.

[14]Isoe-Wada K, Maeda M, Yong J, et al. Positive association between an estrogen receptor gene polymorphism and Parkinson’s disease with dementia. Eur J Neurol. 1999;6(4):431-435.

[15]Wang J, Nuite M, McAlindon TE. Association of estrogen and aromatase gene polymorphisms with systemic lupus erythematosus. Lupus. 2010;19(6):734-740.

[16]Ma SL, Tang NL, Tam CW, et al. Polymorphisms of the estrogen receptor alpha (ESR1) gene and the risk of Alzheimer's disease in a southern Chinese community. Int Psychogeriatr. 2009;21(5):977-986.

[17]Niino M, Kikuchi S, Fukazawa T, et al. Estrogen receptor gene polymorphism in Japanese patients with multiple sclerosis.J Neurol Sci. 2000;179(S1-2):70-75.

[18]Kikuchi S, Fukazawa T, Niino M, et al. Estrogen receptor gene polymorphism and multiple sclerosis in Japanese patients:interaction with HLA-DRB1＊1501 and disease modulation.J Neuroimmunol. 2002;128(1-2):77-81.

[19]Mattila KM, Luomala M, Lehtimki T. Interation between ESR1 and HLA-DR2 may contribute to the development of MS in women.Neurology. 2001;56(9):1246-1247.

[20]Savettieri G, Cittadella R, Valentino P, et al. Lack of association between estrogen receptor 1 gene polymorphisms and multiple sclerosis in southern Italy in humans. Neurosci Lett. 2002;327(2):115-118.

[21]TajouriL, Fernandez F, Tajouri S, et al. Allelic variation investigation of the estrogen receptor with in an Australian multi le sclerosis population. Neurol Sci. 2007;252(1):9-12.

[22]Liu H, Liu ZH, Chen ZH, et al. Distribution of estrogen receptor gene polymorphism in chinese. Shenzangbing yu Touxi Shenyizhi Zazhi. 1999;8(2):105-108.

[23]Castotagnoli A, Maestri I, Bernardi F, et al. PvuII RFLP inside the human estrogen receptor gene. Nucleic Acid Res. 1987;15(2):866-871.

[24]Ushiyama T, Ueyama H , Inoue K, et al. Estrogen receptor gene polymorphism and generalized osteoarthritis. J Rhenmatol. 1998;25(1):134-137.

[25]Carling T, Rastad J, Kindmark A, et al. Estrogen receptor gene polymorphism in postmenopausal primary hyperparathyroidism.Surgery. 1997;122(6):1101-1105.

[26]Polman CH, Reingold SC, Edan G, et al. Diagnostic criteria for multiple sclerosis:2005 revisions to the “McDonald Criteria”. Ann Neurol. 2005;58(6):840-846.

[27]Kurtzke JF. Rating neurologic impairment in multiple sclerosis: an expanded disability status scale (EDSS). Neurology. 1983;33(11):1444-1452.

[28]State Council of the People’s Republic of China. Administrative Regulations on Medical Institution. 1994.