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T polymorphism in Korean vitiligo patients
Journal of Dermatological Science (2004) 35, 181—186
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Association of estrogen receptor 1 intron 1 C/T polymorphism in Korean vitiligo patients Sheng-Yu Jina, Hyun-Ho Parkb, Guang-Zhe Lia, Hee Jae Leea, Mee-Suk Honga, Hae-Jeong Parka, Hun-Kuk Parkc, Jung-Chul Seod, Sung-Vin Yima, Joo-Ho Chunga, Mu-Hyoung Leeb,* a
Department of Pharmacology, College of Medicine, Kohwang Medical Research Institute, Kyung Hee University, Seoul 130-701, South Korea b Department of Dermatology, College of Medicine, Kyung Hee University, Seoul 130-701, South Korea c Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul 130-701, South Korea d Department of Acupuncture and Moxibustion, Gumi Oriental Medical hospital, Daegu Haany University, Gumi, Kyungsangbuk-do, 730-090, South Korea Received 27 November 2003; received in revised form 24 May 2004; accepted 15 June 2004
KEYWORDS Vitiligo; Estrogen receptor 1; Polymorphism
Summary Background: Vitiligo is a common disease characterized by cutaneous white maculae due to loss of melanocytes. It is a polygenic disease, however, the exact pathogenesis of vitiligo is not yet known. The estrogen receptor (ESR) 1 gene was selected as a candidate gene because some researchers treated vitiligo successfully with the steroid-thyroid hormone mixture containing estrogen. Furthermore ESR was expressed in the melanocytes which have an important role in the pigmentation. The polymorphisms of ESR1 gene in vitiligo patients was not reported yet. Objective: To determine whether polymorphisms of ESR1 gene were associated with susceptibility to vitiligo patients in Korean population. Methods: We conducted case—control association study of vitiligo patients (120) and healthy controls (254). Genotypes of ESR1 gene (intron 1 C/T, exon 4 C/G, and exon 8 A/G) were determined by polymerase chain reaction followed by restriction enzyme digestion. Results: Intron 1 T/C allele frequency was significantly different between patients and controls (P = 0.034). Intron 1 T/C genotype distribution (P = 0.021) and allele frequency (P = 0.013) were different between female vitiligo patients and female controls. Intron 1 T/C allele frequency showed significantly difference between generalized type of vitiligo patients and controls (P = 0.044). Genotype distributions and allele frequencies of exon 4 C/G and exon 8 A/G polymorphisms were not different between patients and controls.
* Corresponding author. Tel.: +82 2 958 8512; fax: +82 2 969 6538. E-mail address: [email protected] (M.-H. Lee). 0923-1811/$30.00 # 2004 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2004.06.008
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S.Y. Jin et al.
Conclusion: The present study suggests that ESR1 may be a possible risk factor for female or generalized type of vitiligo patients. # 2004 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
1. Introduction Vitiligo is a skin disorder characterized by loss of melanocytes. It is affecting about 1% of the world’s population [1]. Vitiligo is a multifactorial disorder related with genetic, environmental, local, and endocrine factors. There are many theories about the etiology of vitiligo, including the self-destruct, biochemical, neural, autoimmune, and genetic hypotheses [2]. The causes of vitiligo were focused on three different mechanisms: autoimmune, autocytotoxic, and neural [1,2]. A number of candidate genes have been suggested to mediate susceptibility to vitiligo, including autoimmune regulator (AIRE) [3], cytotoxic T lymphocyte-associated 4 (CTLA4) [4], GTP cyclohydrolase 1 (GCH1) [5], major histocompatibility complex (MHC) [6], vitiligo-associated protein 1 (VIT1) [7], catalase [8], catechol-Omethyltransferase (COMT) [9], angiotensin-converting enzyme (ACE) [10], and systemic lupus erythematosus vitiligo-related (SLEV1) [11], autoimmunity susceptibility locus (AIS1) [12], transporter associated with antigen processing (TAP)/large multifunctional protease (LMP) [13]. Based on all of these observations, vitiligo may be considered as a polygenic disease. It was reported that high estrogen level in the serum is associated with increased skin pigmentation [14]. Estrogen appears to increase the pigment cell activity and suppress the sebaceous gland activity [12]. Some studies stated successful treatment of vitiligo with the steroid-thyroid hormone mixture containing estrogen [15—17]. Human ESR has two isoforms: ESR1 (ESRa) and ESR2 (ESRb). The ESR1 is a ligand-activated transcription factor composed of several domains which are important for hormone binding, DNA binding, and activation of transcrip-
tion [18]. These receptors are known as members of the steroid-thyroid hormone superfamily of nuclear receptors [19,20]. The ESR gene is larger than 140 kb, contains 8 exons, and is located on chromosome 6q25.1 in humans [21,22]. The coding region has a length of 1785 nucleotides and is translated into a protein of 595 amino acids and 66 kDa in mass [23]. This is the first paper to analyze the association of the ESR1 gene polymorphism and vitiligo. We examined genotype distributions and allele frequencies of ESR1 gene, which may be involved in vitiligo.
2. Materials and methods 2.1. Patients and healthy control populations The 120 Korean vitiligo patients (51 men and 69 women) were examined at dermatology clinic in Kyung Hee University Medical Center. Healthy subjects were selected from those who underwent the 2002 health examinations. The 254 (102 men and 152 women) controls did not show clinical evidence of vitiligo or any other disorders (Table 1). This study was approved by the ethics review committee of the Medical Research Institute, Kyung Hee University Medical Center.
2.2. ESR1 gene polymorphisms analysis Three single nucleotide polymorphisms (SNP) were identified in ESR1 intron 1 T/C (NCBI, dbSNP number, rs2234693), exon 4 C/G (NCBI, dbSNP number, rs1801132; codon 325) and exon 8 A/G (NCBI,
Table 1 Clinical characteristics of the 120 Korean vitiligo patients and 254 healthy controls Average age (year, mean S.D.) Number of female/male Onset age (year, mean S.D.) Early onset/late onset Clinical type (generalized/focal) Female (generalized/focal) Male (generalized/focal) With/without other autoimmune diseases
Vitiligo (n = 120)
Healthy controls (n = 254)
34.5 17.9 69 (0.57)/51 (0.43) 27.7 18.8 37 (0.31)/83 (0.69) 53 (0.44)/67 (0.56) 29 (0.42)/40 (0.58) 24 (0.47)/27 (0.53) 9 (0.07)/111 (0.93)
36.3 17.2 152 (0.60)/102 (0.40)
Association of estrogen receptor 1 intron 1 C/T polymorphism in Korean vitiligo patients
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Table 2 Sequences of primers used for ESR1 genotyping Polymorphism site
Primer sets
Annealing temperature (8C)
Restriction enzyme
Allele size (bp)
Intron 1 C/T
50 -ctgccaccctatctgtatcttttcctattctcc-30 50 -tctttctgccaccctggcgtcgattatcatcga-30 50 -gcccgctcatgatcaaacg-30 50 -aggatcatactcggatagagaat-30 50 -gaggagacggaccaaagccac-30 50 -gccattggtgttggatgcatg-30
64
PvuII
58
HinfI
63
BtgI
T: 936 + 436 C: 1372 C: 119 G: 98 + 21 A: 227 G: 129 + 98
Exon 4 C/G codon 325 Exon 8 G/A codon 594
2.3. Statistical analysis
dbSNP number, rs2228480; codon 594). These three SNP contig position are 56318231 (intron 1 T/C), 56420372 (exon 4 C/G), and 56574945 (exon 8 A/G) respectively. The physical distance between intron 1 T/C and exon 8 A/G is 25,6714 bp. Genomic DNA was isolated from EDTA treated blood samples by NucleoSpin DNA isolation kit (Macherey-Nagel GmbH & Co., Du ˝ren, Germany). Polymerase chain reaction (PCR) amplifications were performed using 50 ng of genomic DNA in a 30 ml reaction volume that contained, 2.5 mM dNTP 0.5 ml, 2 U of Taq DNA polymerase (Neruotics Inc, Korea), and PCR primer sets. PCR primers used for ESR1 intron 1 C/T [24], exon 4 C/G, and exon 8 G/A [25] genotyping in this study are listed in Table 2. DNA samples were subjected to 35 cycles of PCR amplification in Mastercycler1 gradient (Eppendorf, Hambug, Germany). The PCR products were digested with the restriction enzyme PvuII, HinfI, and BtgI (New England Biolabs, Beverly, MA, USA), then restriction fragments were separated by agarose gel electrophoresis and ethidium bromide staining.
For case—control association study, we used standard chi-square (x2) test in order to determine the differences in allele or genotype frequencies between vitiligo patients and control populations (Table 3). Estimate haplotype frequencies (EH) program [26] was used to investigate the haplotype relative risk. P values less than 0.05 were considered as statistically significant. To measure deviations from Hardy—Weinberg equilibrium at each polymorphic locus Wright’s [27] F-statistics were calculated using Weir and Cockerham’s [28] multilocus estimators.
3. Results As shown in Table 1, three single nucleotide polymorphisms (SNP) were identified in ESR1 intron 1 T/C, exon 4 C/G and exon 8 A/G. When comparing allelic frequencies of the intron 1 T/C polymorphism
Table 3 Distribution of genotypes and alleles of ESR1 in vitiligo patients and control populations Groups
ESR1 genotypes
ESR1 alleles
Vitiligo Control P-value (n = 120, %) (n = 254, %) PvuII TT (Intron1 T/C) CT CC
46 (38.3)
120 (47.2)
56 (46.7) 18 (15.0)
113 (44.5) 21 (8.30)
HinfI CC 76 (63.3) (Exon 4 C/G) CG 40 (33.3) GG 4 (3.30)
148 (58.3)
BtgI (Exon 8 G/A)
178 (70.1)
GG 90 (75.0) GA 24 (20.0) AA 6 (5.00)
a
Control (%)
P-value
Odds ratio (95% CI)
x2 = 5.08, P = 0.08
T 148 (61.7) 353 (69.5) x2 = 4.50, P = 0.034 C 92 (38.3) 155 (30.5)
1.41 (1.03—1.95)
x2 = 2.06, P = 0.36
A 192 (81.1) 385 (80.3) x2 = 1.64, P = 0.20 G 48 (18.9) 123 (19.7)
0.78 (0.54—1.14)
x2 = 2.07, P = 0.35
G 204 (74.5) 423 (80.1) x2 = 0.36, P = 0.55 A 36 (25.5) 85 (19.9)
0.88 (0.57—1.34)
89 (35.0) 17 (6.70)
67 (26.4) 9 (3.50)
Vitiligo (%)
b
CI: confidence interval. a Control vs. patients using the x2-test with 3 2 contingency table. b Control vs. patients using the x2-test with 2 2 contingency table.
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Table 4 Comparison between vitiligo subtypes and controls Clinical subtypes
Genotype distributionsa Intron 1 TT
Allele frequenciesb Exon 4
TC
CC
CC
Exon 8 CG
GG
GG
Intron 1 GA
AA
T
Exon 4 C
C
Exon 8 G
G
A
Focal
26 33 8 (38.8) (49.3) (11.9) P = 0.39; x2 = 7.72
43 20 4 (64.2) (29.8) (6.00) P = 0.68; x2 = 0.77
50 13 4 (74.6) (19.4) (5.97) P = 0.38; x2 = 1.96
85 49 (63.4) (36.6) P = 0.18, 1.31 (0.88—1.96)
106 28 (79.1) (20.9) P = 0.42, 0.83 (0.52—1.31)
113 21 (84.3) (15.7) P = 0.77, 1.08 (0.64—1.82)
Generalized
20 23 10 (37.7) (43.4) (18.9) P = 0.021; x2 = 1.88
33 20 0 (62.3) (37.7) (0.00) P = 0.15; x2 = 3.76
40 11 2 (75.5) (20.7) (3.80) P = 0.69; x2 = 0.73
63 43 (59.3) (40.7) P = 0.04, 1.55 (1.01—2.39)
86 20 (81.1) (18.9) P = 0.24, 0.73 (0.43—1.23)
91 15 (85.8) (14.2) P = 0.51, 1.22 (0.67—2.21)
Female
23 41 5 (33.3) (59.4) (7.25) P = 0.056; x2 = 5.76
47 19 3 (68.1) (27.5) (4.35) P = 0.35; x2 = 2.09
52 14 3 (75.4) (20.3) (4.35) P = 0.30; x2 = 2.39
87 51 (63.0) (37.0) P = 0.01, 1.73 (1.22-2.66)
113 25 (81.9) (18.1) P = 0.17, 0.70 (0.42—2.66)
118 20 (85.5) (14.5) P = 0.24, 1.39 (0.80—2.42)
Male
23 15 13 (45.1) (29.4) (25.5) P = 0.13; x2 = 4.10
29 21 1 (56.9) (41.2) (1.96) P = 0.078; x2 = 5.11
38 10 3 (74.5) (19.6) (5.88) P = 0.68; x2 = 0.78
61 41 (59.8) (40.2) P = 0.74, 1.0 (0.67—1.77)
79 23 (77.5) (22.5) P = 0.70, 1.12 (0.63—1.96)
86 16 (84.3) (15.7) P = 0.56, 0.82 (0.42—1.60)
Without autoimmune disease
44 50 17 (39.6) (45.1) (15.3) P = 0.096; x2 = 4.68
69 38 4 (62.2) (34.2) (3.60) P = 0.47; x2 = 1.49
81 24 6 (73.0) (21.6) (5.41) P = 0.49; x2 = 1.49
138 84 (62.2) (37.8) P = 0.05, 1.39 (1.00—1.93)
176 46 (79.3) (20.7) P = 0.30, 0.82 (0.56—1.20)
186 36 (83.8) (16.2) P = 0.86, 1.04 (0.68—1.59)
S.Y. Jin et al.
CI: confidence interval. a Control vs. patients using the x2-test with 3 2 contingency table. b Control vs. patients using the x2-test with 2 2 contingency table.
Association of estrogen receptor 1 intron 1 C/T polymorphism in Korean vitiligo patients
between vitiligo patients and controls, significant differences were observed (P = 0.034, OR: 1.42, 95% CI: 1.03—1.95). There were no evidence for a deviation from the Hardy—Weinberg equilibrium in healthy controls (intron 1, P = 0.74; exon 4, P = 0.77; exon 8, P = 0.69, respectively) and vitiligo patients (intron 1, P = 0.99; exon 4, P = 0.90; exon 8, P = 0.06, respectively). When comparing the allelic frequencies of the intron 1 T/C polymorphism between generalized type (P = 0.044, OR: 1.55, 95% CI: 1.01—2.39) vitiligo patients and healthy controls, significant differences were observed (Table 4). In this study, significant differences in intron 1 T/C (P = 0.012, OR: 1.73, 95% CI: 1.12— 2.66) allelic frequencies were observed upon comparison of female vitiligo patients and female controls, but not in ESR1 gene exon 4 C/G and exon 8 A/ G polymorphism (Table 4).
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frequency were significantly different between female healthy controls and female vitiligo patients. Three ESR 1 polymorphisms were investigated using EH program, the haplotype of ESR1 gene was not different (x2 = 13.02, d.f. = 7, P = 0.072). As for genetic diversity, levels of inbreeding within subpopulations and in the total populations were not significant as indicated by jackknifed FIS and FIT estimates calculated over loci (0.019 and 0.023, respectively). In conclusion, this is the first report to suggest the association of polymorphism on intron 1 of ESR1 in vitiligo patients. ESR1 intron 1 C/T polymorphism was associated with female or generalized vitiligo patients. ESR1 gene may be a possible risk factor for the female or generalized type vitiligo patients.
Acknowledgments 4. Discussion Vitiligo is a common skin disease characterized by depigmented patches due to selective destruction of melanocytes. The etiology of vitiligo is unknown, but there are several hypotheses. Vitiligo is a complex genetic disease that may involve genes expressed in melanocytes [1,29,30]. We used a candidate gene approach to study vitiligo susceptibility, and reported the genetic relationship between vitiligo and the ESR1 gene. Ohtake et al. [31] concluded that estrogen receptor-mediated signaling is modulated by a coregulatory-like function of activated a heterodimer of the dioxin receptor (AhR)/aryl hydrocarbon receptor nuclear translocator (ARNT). It is well known that estrogen increases the pigment cell activity [14]. It was reported that human melanocytes express ESR which have specific action in human pigmentation [32,33]. However, the genetic mechanisms of estrogen in human skin pigmentation are not known largely. Suzuki et al. [34] found that the ESR1 intron 1 C/T genotype was associated with familial prostate carcinoma in a Japanese population. Some studies indicated that ESR associated with breast cancer [35,36]. It was reported that the genotype distribution of the ESR1 gene polymorphism is different in various race and ethnic groups [34,37,38]. In our study, the frequency of T (70.5%) and C (29.5%) allele and the distributions rate of TT (47.2%), CT (44.5%), and TT (8.3%) in the control subjects are similar to the previously reported distribution in Chinese populations [38]. Our results revealed that intron 1 C/T allele frequencies were significantly different between generalized type of vitiligo patients and controls. ESR 1 intron 1 C/T genotype distribution and allele
This study was supported by a Biomedical Brain Research Center grant from the Ministry of Health and Welfare of the Republic of Korea (01-PJ8-PG601NE01-0003).
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[26] Terwilliger JD, Ott J. Linkage disequilibrium between alleles at marker loci. In: Handbook of human genetic linkage, 1st. ed. Baltimore: Johns Hopkins University; 1994. p. 188—98. [27] Wright S. The interpretation of population structure by Fstatistics with special regard to systenis of mating. Evolution 1965;19:399—420. [28] Cockerham CC, Weir BS. Covariances of relatives stemming from a population undergoing mixed self and random mating. Biometrics 1984;1:157—64. [29] Chakraborty DP, Roy S, Chakraborty AK. Vitiligo, psoralen, and melanogenesis: some observations and understanding. Pigment Cell Res 1996;9:107—16. [30] Park YK, Kim NS, Hann SK, Im S. Identification of autoantibody to melanocytes and characterization of vitiligo antigen in vitiligo patients. J Dermatol Sci 1996;11:111—20. [31] Ohtake F, Takeyama K, Matsumoto T, Kitagawa H, Yamamoto Y, Nohara K, et al. Modulation of oestrogen receptor signaling by association with the activated dioxin receptor. Nature 2003;423:545—50. [32] Ellis DL, Wheeland RG, Solomon H. Estrogen and progesterone receptors in melanocytic lesions. Arch Dermatol 1985;121:1282—5. [33] Im S, Lee ES, Kim W, On W, Kim J, Lee M, et al. Donor specific response of estrogen and progesterone on cultured human melanocytes. J Korean Med Sci 2002;17:58—64. [34] Suzuki K, Nakazato H, Matsui H, Koike H, Okugi H, Kashiwagi B, et al. Genetic polymorphisms of estrogen receptor alpha, CYP19, catechol-O-methyltransferase are associated with familial prostate carcinoma risk in a Japanese population. Cancer 2003;98:1411—6. [35] Zuppan P, Hall JM, Lee MK, Ponglikitmongkol M, King MC. Possible linkage of the estrogen receptor gene to breast cancer in a family with late-onset disease. Am J Hum Genet 1991;48:1065—8. [36] McGuire WL, Chamness GC, Fuqua SA. Estrogen receptor variants in clinical breast cancer. Mol Endocrinol 1991;5:1571—7. [37] Ruza E, Sotillo E, Sierrasesumaga L, Azcona C, Patino-Garcia A. Analysis of polymorphisms of the vitamin D receptor, estrogen receptor, and collagen Ia1 genes and their relationship with height in children with bone cancer. J Pediatr Hematol Oncol 2003;25:780—6. [38] Cai Q, Shu XO, Jin F, Dai Q, Wen W, Cheng JR, et al. Genetic polymorphisms in the estrogen receptor a gene and risk of breast cancer: results from the Shanghai Breast cancer study. Cancer Epidemiol Biomarkers Prev 2003;12:853—9.
www.intl.elsevierhealth.com/journals/jods
Association of estrogen receptor 1 intron 1 C/T polymorphism in Korean vitiligo patients Sheng-Yu Jina, Hyun-Ho Parkb, Guang-Zhe Lia, Hee Jae Leea, Mee-Suk Honga, Hae-Jeong Parka, Hun-Kuk Parkc, Jung-Chul Seod, Sung-Vin Yima, Joo-Ho Chunga, Mu-Hyoung Leeb,* a
Department of Pharmacology, College of Medicine, Kohwang Medical Research Institute, Kyung Hee University, Seoul 130-701, South Korea b Department of Dermatology, College of Medicine, Kyung Hee University, Seoul 130-701, South Korea c Department of Biomedical Engineering, College of Medicine, Kyung Hee University, Seoul 130-701, South Korea d Department of Acupuncture and Moxibustion, Gumi Oriental Medical hospital, Daegu Haany University, Gumi, Kyungsangbuk-do, 730-090, South Korea Received 27 November 2003; received in revised form 24 May 2004; accepted 15 June 2004
KEYWORDS Vitiligo; Estrogen receptor 1; Polymorphism
Summary Background: Vitiligo is a common disease characterized by cutaneous white maculae due to loss of melanocytes. It is a polygenic disease, however, the exact pathogenesis of vitiligo is not yet known. The estrogen receptor (ESR) 1 gene was selected as a candidate gene because some researchers treated vitiligo successfully with the steroid-thyroid hormone mixture containing estrogen. Furthermore ESR was expressed in the melanocytes which have an important role in the pigmentation. The polymorphisms of ESR1 gene in vitiligo patients was not reported yet. Objective: To determine whether polymorphisms of ESR1 gene were associated with susceptibility to vitiligo patients in Korean population. Methods: We conducted case—control association study of vitiligo patients (120) and healthy controls (254). Genotypes of ESR1 gene (intron 1 C/T, exon 4 C/G, and exon 8 A/G) were determined by polymerase chain reaction followed by restriction enzyme digestion. Results: Intron 1 T/C allele frequency was significantly different between patients and controls (P = 0.034). Intron 1 T/C genotype distribution (P = 0.021) and allele frequency (P = 0.013) were different between female vitiligo patients and female controls. Intron 1 T/C allele frequency showed significantly difference between generalized type of vitiligo patients and controls (P = 0.044). Genotype distributions and allele frequencies of exon 4 C/G and exon 8 A/G polymorphisms were not different between patients and controls.
* Corresponding author. Tel.: +82 2 958 8512; fax: +82 2 969 6538. E-mail address: [email protected] (M.-H. Lee). 0923-1811/$30.00 # 2004 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology. doi:10.1016/j.jdermsci.2004.06.008
182
S.Y. Jin et al.
Conclusion: The present study suggests that ESR1 may be a possible risk factor for female or generalized type of vitiligo patients. # 2004 Published by Elsevier Ireland Ltd on behalf of Japanese Society for Investigative Dermatology.
1. Introduction Vitiligo is a skin disorder characterized by loss of melanocytes. It is affecting about 1% of the world’s population [1]. Vitiligo is a multifactorial disorder related with genetic, environmental, local, and endocrine factors. There are many theories about the etiology of vitiligo, including the self-destruct, biochemical, neural, autoimmune, and genetic hypotheses [2]. The causes of vitiligo were focused on three different mechanisms: autoimmune, autocytotoxic, and neural [1,2]. A number of candidate genes have been suggested to mediate susceptibility to vitiligo, including autoimmune regulator (AIRE) [3], cytotoxic T lymphocyte-associated 4 (CTLA4) [4], GTP cyclohydrolase 1 (GCH1) [5], major histocompatibility complex (MHC) [6], vitiligo-associated protein 1 (VIT1) [7], catalase [8], catechol-Omethyltransferase (COMT) [9], angiotensin-converting enzyme (ACE) [10], and systemic lupus erythematosus vitiligo-related (SLEV1) [11], autoimmunity susceptibility locus (AIS1) [12], transporter associated with antigen processing (TAP)/large multifunctional protease (LMP) [13]. Based on all of these observations, vitiligo may be considered as a polygenic disease. It was reported that high estrogen level in the serum is associated with increased skin pigmentation [14]. Estrogen appears to increase the pigment cell activity and suppress the sebaceous gland activity [12]. Some studies stated successful treatment of vitiligo with the steroid-thyroid hormone mixture containing estrogen [15—17]. Human ESR has two isoforms: ESR1 (ESRa) and ESR2 (ESRb). The ESR1 is a ligand-activated transcription factor composed of several domains which are important for hormone binding, DNA binding, and activation of transcrip-
tion [18]. These receptors are known as members of the steroid-thyroid hormone superfamily of nuclear receptors [19,20]. The ESR gene is larger than 140 kb, contains 8 exons, and is located on chromosome 6q25.1 in humans [21,22]. The coding region has a length of 1785 nucleotides and is translated into a protein of 595 amino acids and 66 kDa in mass [23]. This is the first paper to analyze the association of the ESR1 gene polymorphism and vitiligo. We examined genotype distributions and allele frequencies of ESR1 gene, which may be involved in vitiligo.
2. Materials and methods 2.1. Patients and healthy control populations The 120 Korean vitiligo patients (51 men and 69 women) were examined at dermatology clinic in Kyung Hee University Medical Center. Healthy subjects were selected from those who underwent the 2002 health examinations. The 254 (102 men and 152 women) controls did not show clinical evidence of vitiligo or any other disorders (Table 1). This study was approved by the ethics review committee of the Medical Research Institute, Kyung Hee University Medical Center.
2.2. ESR1 gene polymorphisms analysis Three single nucleotide polymorphisms (SNP) were identified in ESR1 intron 1 T/C (NCBI, dbSNP number, rs2234693), exon 4 C/G (NCBI, dbSNP number, rs1801132; codon 325) and exon 8 A/G (NCBI,
Table 1 Clinical characteristics of the 120 Korean vitiligo patients and 254 healthy controls Average age (year, mean S.D.) Number of female/male Onset age (year, mean S.D.) Early onset/late onset Clinical type (generalized/focal) Female (generalized/focal) Male (generalized/focal) With/without other autoimmune diseases
Vitiligo (n = 120)
Healthy controls (n = 254)
34.5 17.9 69 (0.57)/51 (0.43) 27.7 18.8 37 (0.31)/83 (0.69) 53 (0.44)/67 (0.56) 29 (0.42)/40 (0.58) 24 (0.47)/27 (0.53) 9 (0.07)/111 (0.93)
36.3 17.2 152 (0.60)/102 (0.40)
Association of estrogen receptor 1 intron 1 C/T polymorphism in Korean vitiligo patients
183
Table 2 Sequences of primers used for ESR1 genotyping Polymorphism site
Primer sets
Annealing temperature (8C)
Restriction enzyme
Allele size (bp)
Intron 1 C/T
50 -ctgccaccctatctgtatcttttcctattctcc-30 50 -tctttctgccaccctggcgtcgattatcatcga-30 50 -gcccgctcatgatcaaacg-30 50 -aggatcatactcggatagagaat-30 50 -gaggagacggaccaaagccac-30 50 -gccattggtgttggatgcatg-30
64
PvuII
58
HinfI
63
BtgI
T: 936 + 436 C: 1372 C: 119 G: 98 + 21 A: 227 G: 129 + 98
Exon 4 C/G codon 325 Exon 8 G/A codon 594
2.3. Statistical analysis
dbSNP number, rs2228480; codon 594). These three SNP contig position are 56318231 (intron 1 T/C), 56420372 (exon 4 C/G), and 56574945 (exon 8 A/G) respectively. The physical distance between intron 1 T/C and exon 8 A/G is 25,6714 bp. Genomic DNA was isolated from EDTA treated blood samples by NucleoSpin DNA isolation kit (Macherey-Nagel GmbH & Co., Du ˝ren, Germany). Polymerase chain reaction (PCR) amplifications were performed using 50 ng of genomic DNA in a 30 ml reaction volume that contained, 2.5 mM dNTP 0.5 ml, 2 U of Taq DNA polymerase (Neruotics Inc, Korea), and PCR primer sets. PCR primers used for ESR1 intron 1 C/T [24], exon 4 C/G, and exon 8 G/A [25] genotyping in this study are listed in Table 2. DNA samples were subjected to 35 cycles of PCR amplification in Mastercycler1 gradient (Eppendorf, Hambug, Germany). The PCR products were digested with the restriction enzyme PvuII, HinfI, and BtgI (New England Biolabs, Beverly, MA, USA), then restriction fragments were separated by agarose gel electrophoresis and ethidium bromide staining.
For case—control association study, we used standard chi-square (x2) test in order to determine the differences in allele or genotype frequencies between vitiligo patients and control populations (Table 3). Estimate haplotype frequencies (EH) program [26] was used to investigate the haplotype relative risk. P values less than 0.05 were considered as statistically significant. To measure deviations from Hardy—Weinberg equilibrium at each polymorphic locus Wright’s [27] F-statistics were calculated using Weir and Cockerham’s [28] multilocus estimators.
3. Results As shown in Table 1, three single nucleotide polymorphisms (SNP) were identified in ESR1 intron 1 T/C, exon 4 C/G and exon 8 A/G. When comparing allelic frequencies of the intron 1 T/C polymorphism
Table 3 Distribution of genotypes and alleles of ESR1 in vitiligo patients and control populations Groups
ESR1 genotypes
ESR1 alleles
Vitiligo Control P-value (n = 120, %) (n = 254, %) PvuII TT (Intron1 T/C) CT CC
46 (38.3)
120 (47.2)
56 (46.7) 18 (15.0)
113 (44.5) 21 (8.30)
HinfI CC 76 (63.3) (Exon 4 C/G) CG 40 (33.3) GG 4 (3.30)
148 (58.3)
BtgI (Exon 8 G/A)
178 (70.1)
GG 90 (75.0) GA 24 (20.0) AA 6 (5.00)
a
Control (%)
P-value
Odds ratio (95% CI)
x2 = 5.08, P = 0.08
T 148 (61.7) 353 (69.5) x2 = 4.50, P = 0.034 C 92 (38.3) 155 (30.5)
1.41 (1.03—1.95)
x2 = 2.06, P = 0.36
A 192 (81.1) 385 (80.3) x2 = 1.64, P = 0.20 G 48 (18.9) 123 (19.7)
0.78 (0.54—1.14)
x2 = 2.07, P = 0.35
G 204 (74.5) 423 (80.1) x2 = 0.36, P = 0.55 A 36 (25.5) 85 (19.9)
0.88 (0.57—1.34)
89 (35.0) 17 (6.70)
67 (26.4) 9 (3.50)
Vitiligo (%)
b
CI: confidence interval. a Control vs. patients using the x2-test with 3 2 contingency table. b Control vs. patients using the x2-test with 2 2 contingency table.
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Table 4 Comparison between vitiligo subtypes and controls Clinical subtypes
Genotype distributionsa Intron 1 TT
Allele frequenciesb Exon 4
TC
CC
CC
Exon 8 CG
GG
GG
Intron 1 GA
AA
T
Exon 4 C
C
Exon 8 G
G
A
Focal
26 33 8 (38.8) (49.3) (11.9) P = 0.39; x2 = 7.72
43 20 4 (64.2) (29.8) (6.00) P = 0.68; x2 = 0.77
50 13 4 (74.6) (19.4) (5.97) P = 0.38; x2 = 1.96
85 49 (63.4) (36.6) P = 0.18, 1.31 (0.88—1.96)
106 28 (79.1) (20.9) P = 0.42, 0.83 (0.52—1.31)
113 21 (84.3) (15.7) P = 0.77, 1.08 (0.64—1.82)
Generalized
20 23 10 (37.7) (43.4) (18.9) P = 0.021; x2 = 1.88
33 20 0 (62.3) (37.7) (0.00) P = 0.15; x2 = 3.76
40 11 2 (75.5) (20.7) (3.80) P = 0.69; x2 = 0.73
63 43 (59.3) (40.7) P = 0.04, 1.55 (1.01—2.39)
86 20 (81.1) (18.9) P = 0.24, 0.73 (0.43—1.23)
91 15 (85.8) (14.2) P = 0.51, 1.22 (0.67—2.21)
Female
23 41 5 (33.3) (59.4) (7.25) P = 0.056; x2 = 5.76
47 19 3 (68.1) (27.5) (4.35) P = 0.35; x2 = 2.09
52 14 3 (75.4) (20.3) (4.35) P = 0.30; x2 = 2.39
87 51 (63.0) (37.0) P = 0.01, 1.73 (1.22-2.66)
113 25 (81.9) (18.1) P = 0.17, 0.70 (0.42—2.66)
118 20 (85.5) (14.5) P = 0.24, 1.39 (0.80—2.42)
Male
23 15 13 (45.1) (29.4) (25.5) P = 0.13; x2 = 4.10
29 21 1 (56.9) (41.2) (1.96) P = 0.078; x2 = 5.11
38 10 3 (74.5) (19.6) (5.88) P = 0.68; x2 = 0.78
61 41 (59.8) (40.2) P = 0.74, 1.0 (0.67—1.77)
79 23 (77.5) (22.5) P = 0.70, 1.12 (0.63—1.96)
86 16 (84.3) (15.7) P = 0.56, 0.82 (0.42—1.60)
Without autoimmune disease
44 50 17 (39.6) (45.1) (15.3) P = 0.096; x2 = 4.68
69 38 4 (62.2) (34.2) (3.60) P = 0.47; x2 = 1.49
81 24 6 (73.0) (21.6) (5.41) P = 0.49; x2 = 1.49
138 84 (62.2) (37.8) P = 0.05, 1.39 (1.00—1.93)
176 46 (79.3) (20.7) P = 0.30, 0.82 (0.56—1.20)
186 36 (83.8) (16.2) P = 0.86, 1.04 (0.68—1.59)
S.Y. Jin et al.
CI: confidence interval. a Control vs. patients using the x2-test with 3 2 contingency table. b Control vs. patients using the x2-test with 2 2 contingency table.
Association of estrogen receptor 1 intron 1 C/T polymorphism in Korean vitiligo patients
between vitiligo patients and controls, significant differences were observed (P = 0.034, OR: 1.42, 95% CI: 1.03—1.95). There were no evidence for a deviation from the Hardy—Weinberg equilibrium in healthy controls (intron 1, P = 0.74; exon 4, P = 0.77; exon 8, P = 0.69, respectively) and vitiligo patients (intron 1, P = 0.99; exon 4, P = 0.90; exon 8, P = 0.06, respectively). When comparing the allelic frequencies of the intron 1 T/C polymorphism between generalized type (P = 0.044, OR: 1.55, 95% CI: 1.01—2.39) vitiligo patients and healthy controls, significant differences were observed (Table 4). In this study, significant differences in intron 1 T/C (P = 0.012, OR: 1.73, 95% CI: 1.12— 2.66) allelic frequencies were observed upon comparison of female vitiligo patients and female controls, but not in ESR1 gene exon 4 C/G and exon 8 A/ G polymorphism (Table 4).
185
frequency were significantly different between female healthy controls and female vitiligo patients. Three ESR 1 polymorphisms were investigated using EH program, the haplotype of ESR1 gene was not different (x2 = 13.02, d.f. = 7, P = 0.072). As for genetic diversity, levels of inbreeding within subpopulations and in the total populations were not significant as indicated by jackknifed FIS and FIT estimates calculated over loci (0.019 and 0.023, respectively). In conclusion, this is the first report to suggest the association of polymorphism on intron 1 of ESR1 in vitiligo patients. ESR1 intron 1 C/T polymorphism was associated with female or generalized vitiligo patients. ESR1 gene may be a possible risk factor for the female or generalized type vitiligo patients.
Acknowledgments 4. Discussion Vitiligo is a common skin disease characterized by depigmented patches due to selective destruction of melanocytes. The etiology of vitiligo is unknown, but there are several hypotheses. Vitiligo is a complex genetic disease that may involve genes expressed in melanocytes [1,29,30]. We used a candidate gene approach to study vitiligo susceptibility, and reported the genetic relationship between vitiligo and the ESR1 gene. Ohtake et al. [31] concluded that estrogen receptor-mediated signaling is modulated by a coregulatory-like function of activated a heterodimer of the dioxin receptor (AhR)/aryl hydrocarbon receptor nuclear translocator (ARNT). It is well known that estrogen increases the pigment cell activity [14]. It was reported that human melanocytes express ESR which have specific action in human pigmentation [32,33]. However, the genetic mechanisms of estrogen in human skin pigmentation are not known largely. Suzuki et al. [34] found that the ESR1 intron 1 C/T genotype was associated with familial prostate carcinoma in a Japanese population. Some studies indicated that ESR associated with breast cancer [35,36]. It was reported that the genotype distribution of the ESR1 gene polymorphism is different in various race and ethnic groups [34,37,38]. In our study, the frequency of T (70.5%) and C (29.5%) allele and the distributions rate of TT (47.2%), CT (44.5%), and TT (8.3%) in the control subjects are similar to the previously reported distribution in Chinese populations [38]. Our results revealed that intron 1 C/T allele frequencies were significantly different between generalized type of vitiligo patients and controls. ESR 1 intron 1 C/T genotype distribution and allele
This study was supported by a Biomedical Brain Research Center grant from the Ministry of Health and Welfare of the Republic of Korea (01-PJ8-PG601NE01-0003).
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