DNA polymorphism in B-domain of the estrogen receptor-a among Japanese women Jiro Fujimoto, Reiko Hirose, Satoshi Ichigo, Hideki Sakaguchi, and Teruhiko Tamaya Department of Obstetrics and Gynecology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City 500, Japan A silent mutation in B-domain of the estrogen receptor-a (ER B) change codon 87 (from GCG to GCC) is clinically correlated with frequent spontaneous abortion and familial history of breast cancer among Caucasian patients. However, none of the 167 Japanese female patients and 46 Japanese female healthy volunteers showed ER B variant. Therefore, this DNA polymorphism might involve a genetic racial difference, and appears not to be correlated with frequent spontaneous abortion or familial history of breast cancer at least among Japanese women. (Steroids 63:146–148, 1998) © 1998 by Elsevier Science Inc. Keywords: DNA polymorphism; B-domain of the estrogen receptor-a; Japanese women
Introduction Sex steroids, especially estrogen, contribute to the development, growth and functional regulation of the female genital tract and the corresponding tumor tissues via steroid receptor cascades.1 Deletion and point mutation in DNA and estrogen binding sites,2 exon splicing variants3 in estrogen receptor-a (ER) in endometrial cancers, and an exon splicing variant4 in ovarian endometriosis have recently been elucidated, but these variants seem unrelated to any clinical significance.5–9 However, the presence of a silent mutation in the ER B region change codon 87 is clinically correlated with frequent spontaneous abortion and familial history of breast cancer among Caucasian patients.10 –13 This prompts us to study the contribution of ER B variant as a DNA polymorphism to diseases of the female genital tract in Japanese women.
from those women and the Research Committee for Human Subjects. Caucasian and Japanese women’s backgrounds are showed in Tables 1 and 2, respectively. Frequent spontaneous abortion was defined that the patient’s obstetrical history involves spontaneous abortion more than three times as habitual abortion.
Isolation of genomic DNA Ten mL of blood was collected into heparin sulfate-containing tubes. The blood in 20 mL Ficoll-Conray solution (IBL, Gumma, Japan) was centrifuged at 400 3 g for 30 min. The layer for mononuclear cells in the solution was aspirated, and the cells were washed with phosphate buffer three times. The cells in 500 mL TE buffer (10 mM Tris-HCl, pH 8.0 and 1 mM ethylenediamine tetraacetic acid, pH 8.0) were incubated with 0.1 mg/mL proteinase K and 0.5% sodium dodecyl sulfate (SDS) at 55°C for 90 min. The genomic DNA was extracted with phenol-chloroform, and precipitated with 0.3 M sodium acetate and ethanol.
Patients and methods
Polymerase chain reaction (PCR)
Patients
PCR for ER gene was carried out with the extracted genomic DNA (1 mg) and 0.1 mM specific primers using the IWAKI thermal sequencer TSR-300 (Iwaki Glass, Tokyo, Japan), with Vent DNA polymerase (New England Biolabs, Beverly, Massachusetts, USA) in 10 mM KCl, 20 mM Tris-HCl, pH 8.8, 10 mM (NH4)2SO4, 2 mM MgSO4, 0.1% Triton X-100, and 0.15 mM deoxynucleotide phosphates for 25 cycles, consisting of 1 min at 94°C for denaturation, 1 min at 55°C for annealing, and 1 min at 72°C for extension. The oligodeoxynucleotides of specific primers in PCR were synthesized according to the published information on ER gene (14) as follows: sense primer: 59-AACGCGCAGGTCTACGGTCA-39 (437– 456); antisense primer: 59-TCGGAGACACGCTGTTGAGT-39 (529 –548).
One hundred and sixty-seven Japanese female patients and 46 Japanese female volunteers, ranging from 16 to 77 years of age, and 18 Caucasian female volunteers provided 10 mL of peripheral blood at the Department of Obstetrics and Gynecology, Gifu University School of Medicine, between January 1995 and October 1997. Agreements for the following studies were obtained
Address reprint requests to Jiro Fujimoto, M.D., Ph.D., Department of Obstetrics and Gynecology, Gifu University School of Medicine, 40 Tsukasa-machi, Gifu City 500, Japan. Received August 12, 1997; accepted November 13, 1997. Steroids 63:146 –148, 1998 © 1998 by Elsevier Science Inc. All rights reserved. 655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00 PII S0039-128X(97)00157-8
DNA polymorphism in estrogen receptor gene: Fujimoto et al.
Figure 1 Identification of ER B genotype. Standard ER B, antisense oligonucleotide for ER B (wild type) gene, Standard ER B9, antisense oligonucleotide for ER B9 (variant) gene, ER B probe, biotinylated sense oligodeoxynucleotide for ER B gene, ER B9 probe, biotinylated sense oligodeoxynucleotide for ER B9 gene. Table 1 Backgrounds of Caucasian Women as Controls Nationality
No. of cases
Age (years)
6(2) 4(1) 4 2 2 18
31–59 26–57 19–28 23–42 44–52 19–59
USA Canada Australia UK Spain Total
Numbers in parentheses: no. of patients with ER BB9 heterozygous genotype.
B probe, and ER BB9 was positive for both ERs as the same intensity bands (Figure 1). The ER B-variant analyses were done in 18 healthy Caucasian female volunteers as positive controls (Table 1), and were studied in 167 Japanese female patients and 46 Japanese female healthy volunteers as the test group (Table 2). In Southern blots for the six Caucasian subjects shown, Subjects 4 and 5 revealed ER BB9, and the others ER BB (Figure 2). Integrally, three cases were ER BB9, and the other 15 ER BB. On the other hand, in the six Japanese cases shown, all revealed ER BB (Figure 3). Integrally, all subjects among the 167 Japanese female patients and 46 Japanese female healthy volunteers demonstrated ER BB.
Southern blot analysis PCR products with standard oligonucleotides complemented with ER B or ER B9 genes were applied to agarose gel for electrophoresis performed at 50 –100 V. PCR products were capillarytransferred to an Immobilon transfer membrane (Millipore Corp., Bedford, Massachusetts, USA) for 16 h. The membrane was dried at 80°C for 30 min, and was UV-irradiated to tightly fix PCR products. PCR products on the membrane were prehybridized in 1 M NaCl, 50 mM Tris-HCl, pH 7.6, and 1% SDS at 42°C for 1 h, and then hybridized in the same solution with the biotinylated oligodeoxynucleotide probes synthesized from the sequences of ER gene including codon 87 between the specific primers at 65°C overnight. Specific bands hybridized with the biotinylated probes were detected with Plex Luminescent Kits (Millipore Corp.). The oligodeoxynucleotides as biotinylated specific probes and the standard oligonucleotides for the Southern blot analyses were synthesized as follows: ER B probe: 59-TCTGAGGCT GCG *GCGTTCGG-39 ER B9 probe: 59-TCTGAGGCT GCC *GCGTTCGG-39 ER B standard: 39-AGACTCCGA CGC *CGCAAGCC-59 ER B9 standard: 39-AGACTCCGA CGG *CGCAAGCC-59 *was assigned with aberrant nucleotide in codon 87.
Results For identification of ER genotype (wild type, ER BB; variant heterozygotes, ER BB9; and variant homozygotes, ER B9B9), two types of Southern blot analyses using ER B and ER B9 probes were carried out. The same amount of standard ER B and ER B9 could be shown as the same intensity of the bands between the two Southern blots. ER BB showed positive for ER B probe and negative for ER B9 probe. On the other hand, ER B9B9 showed positive for ER B9 probe and negative for ER
Discussion ER B9 genotype (ER BB9 or ER B9B9) was positive in 10 to 20% of Caucasian women as controls.10 –13 In the present study, ER BB9 genotype was detected in 16.7% (3/18) of Caucasian women. However, none of the 213 Japanese women showed ER B9 variant. Therefore, the DNA polymorphism of ER B region might involve a genetic racial difference, and appears not to be correlated with history of
Table 2 Backgrounds of Japanese Women Disease Endometriosis Hypoplastic uterus Habitual abortion Breast cancer PCO Endometrial cancer Ovarian cancer RKH Pituitary anovulation Uterine cervical cancer Vulvar cancer Kallmann syndrome Controls Total
No. of cases
Age (years)
46 32 30 30 25 18 6 4 5 2 1 1 46
20–45 19–35 26–68 33–63 18–34 34–69 34–57 17–28 17–37 41–47 77 16 16–77
246
16–77
Overlap of diseases is counted. PCO, polycystic ovarian syndrome; RKH, Rokitansky-Ku¨sterHauser syndrome. (n 5 213).
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Papers
Figure 2 ER B genotype in Caucasian women as positive controls. Caucasian women Cases 4 and 5 showed ER BB9, and the others ER BB.
Figure 3 ER B genotype in Japanese women. All Japanese women showed ER BB.
frequent spontaneous abortion or familial history of breast cancer at least among Japanese women. The incidence of frequent spontaneous abortion is approximately 0.3% per pregnancy.15 It is difficult to say whether differences in the frequency of genetic habitual abortion are present among the human races. On the other hand, the frequency of breast cancer in Caucasian and Japanese women is approximately 17 to 25 and 3.5 per 100,000 population, respectively.16 The great difference might be derived from nutritional and/or racial factors, and ER B gene polymorphism might give us one of the keys to the geographic cline of breast cancer. Genetic linkage between gene polymorphisms in estrogen receptor and familial diseases in the various human races needs to be clarified in the future.
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