Hum. Reprod. Advance Access originally published online on October 6, 2005
Human Reproduction 2006 21(2):554-557; doi:10.1093/humrep/dei326
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Combined estrogen receptor 
and estrogen receptor 
genotypes influence the age of menarche
1 Department of Endocrinology and 2 Laboratory of Reproductive Genetics, University of Ioannina, 45110 Ioannina, Greece
3 To whom correspondence should be addressed. E-mail: atsatsou{at}cc.uoi.gr
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Abstract |
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BACKGROUND: Age at menarche has a strong genetic influence. We reported recently an association between the XbaI (351A
C)and PvuII (397TC) polymorphisms of the estrogen receptor (ER)
gene with the age of menarche in Greek adolescents. In the present study, we examined whether ER
genotypes alone, or in combination with ER genotypes, may also influence onset of menarche. METHODS: We performed genotyping for the single nucleotide polymorphisms 1730AG and 1082GA of the ER gene and examined their association with the age of menarche in the same cohort of 145 Greek girls. We then looked for a possible effect of combined ER and genotypes on the age of menarche. RESULTS: Menarche occurred 7 months later in girls with the AA genotype of the 1730AG polymorphism than in girls with the AG genotype (mean ± SD: 13.23 ± 1.24 versus 12.66 ± 1.26 years, respectively; P = 0.005). The 1082GA polymorphism was not detected in any of the girls examined. A significant effect of combined ER and genotypes was also apparent. Menarche occurred 11 months later in girls bearing the AA/TT,AA (ER, ER) genotypes compared with girls with the CC/CC,AG genotype (13.30 ± 1.27 nersus 12.41 ± 1.28 years; P = 0.042). The difference remained significant after adjusting for body mass index (P = 0.034). CONCLUSION: Combined ER and ER polymorphisms may influence the age of menarche.
Key words:
estrogen receptor /estrogen receptor /genotypes/genetic polymorphisms/menarche
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Menarche depends on the maturation and co-ordination of the hypothalamic–pituitary–ovarian axis with the female reproductive system and other endocrine organs, including the adipose tissue (Carr, 1998
). The timing of menarche is regulated by a variety of environmental and genetic factors. Family and twin studies have indicated that the genetic contribution may be more important than environmental effects, since 53–74% of the variation in age of menarche can be attributed to genetic factors (Sharma, 1983, van den Akker et al., 1987, Treloar and Martin, 1990, Kaprio et al., 1995). However, the specific genes involved in this event are not yet well defined.
More than 30 years ago, Frisch and McArthur (1974) proposed that a given amount of body fat is necessary before the onset of menstrual cycles. This claim was recently substantiated by establishing that leptin constitutes the permitting signal, informing the brain on the amount of energy stored in adipose tissue (Kiess et al., 2000; Mantzoros, 2000) and that a polymorphic variant of the leptin gene may influence the onset of menarche in interaction with maternal age (Comings et al., 2001).
Ovarian estrogens appear to play an important role in the differentiation, maturation and function of the reproductive system—and also, in females, the distribution of adipose tissue—through endocrine and paracrine effects mediated by the activation of estrogen receptors (ER) (Enmark and Gustafsson, 1999). Two such receptors have been identified, ER and ER. Both subtypes have been found in the female reproductive organs with overlapping but not identical tissue distribution, and with different or complementary contribution to reproductive functions (Kuiper et al., 1996, Enmark et al., 1997).
Polymorphic variants of both ER and ER genes have been identified in recent years and studied for possible association with reproductive and other clinical outcomes (Georgiou et al., 1999; Syrrou et al., 1999; Weel et al., 1999). Such allelic variants could also account for the genetic variability in the age of menarche. Indeed, we have recently shown that two polymorphisms of the ER gene, in particular XbaI (351AC) and PvuII (397TC) may influence the age of menarche in healthy adolescent Greek girls (Stavrou et al., 2002).
In the present study, we examined the association of 1082GA and 1730AG polymorphisms of the ER gene with the age of menarche and their potential interaction with ER genotypes in influencing this event in the same study population.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Subjects
The study population consisted of 145 healthy adolescent girls from a closed rural community in northwest Greece as described previously (Stavrou et al., 2002
). This homogeneous population was selected with the anticipation that environmental and cultural heterogeneity, which could possibly create some variability in the age of menarche, would be minimal. Information on the age of menarche was taken through personal interviews with the adolescents and their mothers. Informed consent of the girls and their parents, and approval of the study by the University Hospital of Ioannina Ethics Committee were obtained.
Genotyping
Details on genomic DNA extraction and genotyping for the XbaI (351AC) and PvuII (397TC) polymorphisms (Herrington and Howard, 2003) of the ER gene have been described previously (Stavrou et al., 2002).
Genotyping for the 1082GA (Sundarrajan et al., 2001) and 1730AG (Kealey et al., 2001) polymorphic variants of the ER gene was carried out as follows. Specific DNA amplification was performed by PCR using 1 unit of recombinant Taq DNA polymerase (Gibco BRL, Göteborg, Sweden) in DNA thermocycler PTC-100 (Peltier-Effect Cycling, MJ Research, Watertown, Massachusetts, USA). The ligand binding domain of exon 5 and the 3'-untranslated region of exon 8 of the ER gene were amplified using the following primer pairs: 5'-TCTTGCTTTCCCCAGGCTTT-3', 5'-ACCTGTCCAGAA CAAGATCT-3' and 5'-GACCTGCTGCTGGAGATGCT-3', 5'-AAT GAGGGACCACACAGCA-3', respectively.
PCR products were analysed for RFLP using RsaI and AluI restriction enzymes (Gibco BRL). Nucleotide exchange G–A at nucleotide 1082 in exon 5 created a recognition site for RsaI, and exchange AG at nucleotide 1730 in the 3' untranslated region of exon 8 introduced a recognition site for AluI (Fig. 1). Enzyme digestion products underwent electrophoresis on 2% agarose gel and the separation patterns were photographed under ultraviolet illumination. The resulting genotypes for RsaI (1082GA) and AluI (1730 AG) polymorphic sites were characterized as GG, GA, AA and AA, AG, GG, respectively.
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To meet quality control standards and avoid genotyping errors, all samples were run in duplicate and read by two investigators independently. In addition, genotyping was repeated in 20% of randomly selected samples. There was full agreement between the two investigators reading the gels.
Statistical analysis
The t-test and one-way analysis of variance (ANOVA) were used for comparison conducted in Advanced Statistics Package for Social Sciences (SPSS Inc., Chicago, IL, USA). All P-values are two-tailed and P < 0.05 was considered significant.
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Results |
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Association of ER
genotypes with the age of menarcheThe overall mean ( ± SD) age at menarche was 12.92 ± 1.26 years; this is a figure typical for the rural population of this region. Of note, heterozygotes for the ER
1730AG polymorphism were over-represented, whereas homozygotes for the presence of the polymorphic site were not detected, deviating from Hardy–Weinberg equilibrium (P = 0.005). Regarding the ER 1082GA polymorphism, only one genotype (GG) was present in our population study. A similar deviation from Hardy–Weinberg equilibrium was observed for the ER 351AC and 397TC polymorphisms as previously reported by Stavrou et al. (2002). This is highly suggestive of the presence of genetic drift that can be observed in closed communities, as was the case for our study population.
This was further substantiated by comparing the observed allele frequencies (Table I) with allele frequencies derived from other populations. Thus, frequencies in the order of 37– 64% and 94–99% for the A allele of the ER 1730AG and the G allele of the 1082GA polymorphisms, respectively, have been described in other populations (Rosenkranz et al., 1998; Lambert et al., 2001; Arko et al., 2002). With regard to ER 397TC and 351AC polymorphisms, the frequencies for the T and A alleles were 53.4% and 43.4%, respectively, whereas in the general Greek population these are 48% and 41.5% (I.Georgiou, N.Xita, L.Lazaros, unpublished observation).
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Menarche occurred 7 months later in girls with the AA genotype (mean ± SD: 13.23 ± 1.24 years) than in girls with the AG genotype (12.66 ± 1.26 years) of the ER 1730AG polymorphism (P = 0.005).
Effect of combined ER and ER genotypes on the age of menarche
Since ER and ER genes may interact in their biological effects on reproductive functions, we investigated whether combined genotypes of both ER 351AC and 397TC and ER 1730AG polymorphisms may also influence the age of menarche. As shown in Table II, girls bearing the AA/TT haplotype of ER polymorphisms in combination with the AA genotype of ER (Group 1) had 11 months delay in menarche compared with girls bearing the genotype combination CC/CC,AG (Group 2). In addition, menarche occurred 4 months later when girls in Group 1 were compared with girls carrying all other genotype combinations (Group 3). The differences were significant when the groups were compared with each other (P = 0.04). There appeared to be a trend for earlier onset of menarche for girls carrying one or more of the polymorphic C/C,G alleles of ER and ER, respectively—the greater the number of these alleles, the earlier the age of menarche.
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After adjusting for BMI (recorded at the time of evaluation), the difference in menarcheal age between AA homozygotes and the AG heterozygotes for the 1730AG polymorphism was still significant (P = 0.006). Similarly, the difference in age of menarche between the girls with the genotype combination AA, AA/TT in Group 1 compared with the groups 2 and 3 remained the same (P = 0.034).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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Family and twin studies have shown that genetic factors are related to age of menarche. Thus, significant correlations between age at menarche in mothers and daughters along with significant differences in menarcheal age between monozygotic and dizygotic twin pairs have been described (Treloar and Martin, 1990
). We reported recently that age at menarche was associated with the XbaI (351AC) and possibly the PvuII (397TC) polymorphism of the ER gene in healthy adolescent Greek girls (Stavrou et al., 2002). In particular, menarche occurred 6 months later in girls with the AA genotype of the 351 AC polymorphism than in girls with AC or CC genotypes and tended to occur later in TT homozygotes of the 397TC polymorphism than in TC and CC genotype carriers. In the current study, we examined whether there was also an association between ER genotypes and age at menarche in the same study population, since ER receptors are also distributed in reproductive tissues and may influence reproductive function (Kuiper et al., 1997; Hiroi et al., 1999; Sar and Welsch, 1999; Pelletier and El-Alfy, 2000).
The gene encoding ER is located in chromosome 14q 22–24 (Enmark et al., 1997). Five novel polymorphisms have been identified so far within the ER gene (Rosenkranz et al., 1998). Among these, two silent mutations, 1082 GA within exon 5 (RsaI polymorphism) and 1730 AG in the 3'-untranslated region of exon 8 (AluI polymorphism) were selected in the present study, since these polymorphisms have been implicated in other conditions where estrogen exposure is considered to be an important risk modifier (Rosenkranz et al., 1998; Eastwood et al., 2002).
In our study, the 1082GA polymorphic variant was not found in any of the girls examined, indicating that this polymorphism may be rare in Greek or South European populations. With regard to 1730AG polymorphism, however, AA homozygotes appeared to delay menarche by 7 months compared with heterozygotes for the polymorphism indicating that, in addition to ER genotypes, ER genotypes may also contribute to genetic variability of the age of menarche.
In our study, we aimed for a population that would have homogeneity for cultural parameters and environmental influences. The lack of Hardy–Weinberg equilibrium for both ER and ER polymorphisms also suggests that this is a closed community with probably considerable genetic drift due to inbreeding in small communities (Rousset and Raymond, 1995; Vogel and Motulsky, 1997).
Previous studies have shown significant associations of the 1082GA and 1730AG polymorphisms with reproductive dysfunctions (Sundarrajan et al., 2001). In addition, the 1082G A polymorphism has been associated with anorexia nervosa (Rosenkraz et al., 1998; Eastwood et al., 2002).
Although the above polymorphisms do not lead to amino acid changes in the ER protein, it is possible that this polymorphism is in linkage disequilibrium with other regulatory sequence variations that may affect gene expression or function (Yaich et al., 1992). Alternatively, it is believed that single nucleotide polymorphisms—even when situated in the untranslated region—may cause different structural folds of mRNA, and thus influence the expression of the gene (Shen et al., 1999).
The second hypothesis we tested in this study was whether combined genotypes of both ER and ER polymorphisms may also influence the age of menarche. This hypothesis was based on the knowledge that, in addition to homodimers, functional ER/ER heterodimers may interact in their biological effects (Cowley et al., 1997). Indeed, we observed a positive additive effect of combined 351AC, (397TC) (ER) and 1730AG (ER) genotypes on the age of menarche. In particular, the AA/TT haplotype of ER combined with the AA genotype of ER is associated with later onset of menarche by 11 months compared with the genotype combination CC/CC,AG. Furthermore, there appeared to be a trend for earlier menarche for the girls with one or more of the polymorphic C/C,G alleles of the respective genes in that, the higher the number of these alleles, the earlier the onset of menarche—suggesting a dose-response effect. This implies that ER and ER genes may interact in their effects on the age of menarche and this effect may be modified by the presence of certain genotype combinations of both ER subtypes.
Significant interactions between the ER 1730AG polymorphism and the ER 351AC and 397TC polymorphisms were also reported in a study on Alzheimer’s disease in the U.K (Lambert et al., 2001). This study suggested that the risk for Alzheimer’s disease may be modulated by certain ER and ER variants, which influence their expression and/or biological activities.
In conclusion, the findings of the present study suggest that the 1730AG polymorphism of the ER gene is associated with the age of menarche among healthy adolescent girls. In addition, combined genotypes of both ER and ER polymorphisms may further influence menarcheal age, suggesting interaction between these two ER subtypes in affecting this event. Thus, allelic variants of both ER and genes may contribute to the genetic variability in the age of menarche.
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Acknowledgements |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
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We are grateful to Dr T. Trikalinos (Laboratory of Hygiene and Epidemiology, University of Ioannina) for his help and support during statistical analysis.
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Submitted on May 14, 2005; resubmitted on August 13, 2005; accepted on September 9, 2005.
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