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Are CYP1A1, CYP17 and CYP1B1 mutation genes involved on girls with precocious puberty? A pilot study
European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 140–144
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Are CYP1A1, CYP17 and CYP1B1 mutation genes involved on girls with precocious puberty? A pilot study Cezar Noboru Matsuzaki a, Jose´ Maria Soares Ju´nior a,*, Durval Damiani b, Raymundo Soares de Azevedo Neto c, Ka´tia Caˆndido Carvalho a, Felisbela Soares de Holanda a, Eline Maria Stafuzza a, Jose´ Alcione Macedo Almeida a, Edmund Chada Baracat a a
Disciplina de Ginecologia do Departamento de Obstetrı´cia e Ginecologia da Faculdade de Medicina da Universidade de Sa˜o Paulo, 05403000, Sa˜o Paulo, SP, Brazil b Departamento de Pediatria da Faculdade de Medicina da Universidade de Sa˜o Paulo, 05403000, Sa˜o Paulo, SP, Brazil c Departamento de Patologia da Faculdade de Medicina da Universidade de Sa˜o Paulo, 05403000, Sa˜o Paulo, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 June 2014 Received in revised form 23 July 2014 Accepted 29 July 2014
Objectives: To investigate three genes associated with puberty timing in girls with central precocious puberty by evaluating the association between polymorphism in the gene sequence codifying the enzymes participating in steroidogenesis, CYP1A1, CYP17, and CYP1B1 and central precocious puberty. Study design: A total of 177 patients was included and divided into two groups: Case group with 73 girls diagnosed with central precocious puberty; Control group with 104 girls with puberty onset after 8 years of age who were followed at the Sector of Gynecology of Childhood and Adolescence, Division of Gynecology Clinic, HC-FMUSP. Polymorphism presence was assessed in the genes involved in estrogen metabolism (CYP1A1, CYP17, and CYP1B1) by the restriction fragment length polymorphism (RFLP) technique using DNA from peripheral blood. Results: No significant difference in the distribution of the CYP1A1 Mspl (p = 0.86) and CYP17 (p = 0.12) genotypes was detected between the two study groups. As for CYP1B1 Eco571, the mutated C/C genotype was found to be more frequent in the control group than in the case group (p = 0.03). Conclusion: Our data suggest the CYP1B1 Eco571 gene variant is associated with puberty timing. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Genetic polymorphism Estrogens Precocious puberty
Introduction Puberty timing is still unknown. Its mechanism is the source of debate involving genetic susceptibility along with nutritional and environmental factors. The genetic factors influencing puberty timing are yet to be determined [1]. Most studies on the timing of early puberty correlate central factors, which are associated with neurotransmitters, such as glutamate and the kisspeptin/GPR54 complex [2]. There are few studies assessing the peripheral factors which may influence early activation of the hypophyseal–hypothalamic axis. For some researchers, estradiol would be not only the consequence of puberty onset but also a contributory factor in the development of the hypophyseal–hypothalamic axis [3–5]. In a few of the prospective genes, such as those codifying the CYP enzyme complex,
which control the biosynthesis, action, and metabolism of the sex steroids, polymorphisms have been found associated with variations on puberty timing [6]. Such genes are also associated with other gynecological dysfunctions involving estrogen action, such as leiomyoma, endometriosis, and breast cancer [7,8]. The CYP17 gene is believed to be involved in increased production of sex steroids [9]. On the other hand, the CYPA1 and CYPB1 polymorphisms may be related to more active metabolites (4-OH estradiol and 16-OH estradiol) acting on the estrogen receptor and on estrogen-signaling pathways [10]. Therefore, our study aimed at investigating the possibility that the association between such polymorphisms and the central precocious puberty process is brought on by increased estrogen action and attendant GnRH release.
Materials and methods * Corresponding author. Tel.: +55 11 26617838; fax: +55 11 26617838. E-mail address: [email protected] (J.M.S. Ju´nior). http://dx.doi.org/10.1016/j.ejogrb.2014.07.042 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
A total of 177 patients was selected from a group of 1685 patients being treated at the Sector of Gynecology of Childhood and
C.N. Matsuzaki et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 140–144
Adolescence, Gynecology Discipline, Department of Obstetrics and Gynecology, School of Medicine of the University of Sa˜o Paulo (FMUSP). The 177 patients were allocated to two groups: Case group with 73 girls with a current or previous diagnosis of precocious puberty and Control group with 104 girls who showed no signs of puberty until they were 8 years old. The diagnosis of central precocious puberty was based on the clinical features, laboratory exams (LH, FSH, GnRH stimulated gonadotropin levels, estradiol levels), bone age, pelvic ultrasound and brain MRI. The exclusion criteria comprised presence of ovarian or adrenal neoplasms, Cushing syndrome, McCune–Albright syndrome, eye disorders, clinical or laboratory hypothyroidism, use of hormone medications prior to diagnosis, and use of psychotropic or antiepileptic medication. The project was approved by the Ethics Committee for Analysis of Research Projects at HC-FMUSP and FMUSP (CAPPesq–research protocol No. 1120/09). The volunteers were included after their legal guardians freely signed an informed consent statement. Polymorphonuclear leukocytes separated from blood samples were utilized for the patients’ total DNA extraction, carried out with the QIAmp1 DNA mini Blood (Qiagen) kit. The CYP1A1 polymorphism derives from the replacement of cytosine with thymine in the 30 noncoding region, which creates an Mspl restriction site allowing the identification of the following genotypes: CYP1A1wt/wt (wild homozygote), CYP1A1wt/vt (heterozygote), and CYP1A1vt/vt (mutant homozygote) [10]. The 50 region, untranslated from CYP17, becomes polymorphic with the substitution of thymine for cytosine, creating an Mspl restriction site, which identifies two alleles, A1 (wild) and 2 (mutant). The latter increases CYP17 expression, boosting estradiol biosynthesis [9]. Replacing a guanine base with cytosine in exon 3 of the CYP1B1 gene leads to the substitution of leucine for valine in codon 432, creating an Eco 571 restriction site, enabling identification of the G/ G (wild homozygote), G/C (heterozygote), and C/C (mutant homozygote) genotypes [8]. Analysis of the polymorphisms in the CYP1A1, CYP17, and CYP1B1 genes was performed using the PCR-RFLP (restriction fragment length polymorphism) technique. The PCR reactions were produced with 50 ng of total DNA in 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 0.2 mM dNTP, 0.1 mM of each initiator, 1.5 mM of MgSO2, and 0.4 U of Platinum Taq DNA Polymerase High Fidelity (Life Biosciences). The cycling conditions were as follows: preheating to 95 8C for 10 min, followed by 40 cycles at 95 8C for 15 s, at 55 8C for 1 min, at 72 8C for 1 min, followed by a final extension step at 70 8C for 7 min. A specific initiator oligonucleotide was used for each of the polymorphisms that were being investigated. The oligonucleotides are described in Table 1. Following amplification, the PCR fragments corresponding to the CYP1A1, CYP17, and CYP1B1 genes were digested with the restriction enzymes (Promega, Brazil) specific for each polymorphism as reported in the literature (Table 1). Fragments were analyzed in 1% to 3% agarose gel and stained with ethidium
141
bromide. The PCR and RFLP conditions were those already standardized for each gene as described in the literature [8–10]. After data collection, the mean, median, and standard deviation were calculated. Next, sample homogeneity was assessed by the Bartlett test. Results were displayed as mean standard error of the mean. When the two groups were involved in a comparison, the chisquare test was employed. For proportion analysis, the Fisher test was used, and for polymorphisms, a test of Hardy–Weinberg equilibrium. Analyses were carried out with the SPSS version 16.0 software program. In all tests, the significance level for rejecting the null hypothesis was set at 5% or 0.05 (p < 0.05). Results The clinical and epidemiological characteristics of the 73 girls with central precocious puberty (Case group) and of the 104 girls with normal puberty (Control group) making up our study sample are shown in Table 2. At the onset of puberty, the mean age of the girls in the Control group showed they were older (114.9 19.3 months) than those in the Case group (75.8 14.9 months, p < 0.01). Also, at menarche, the mean age of the control group (143.9 15.5 months) was significantly greater than that of the case group (128.7 31.6 months). There was no difference in the mother’s age at menarche between the two groups, i.e., the Control group (147.9 17.7 months) and the Case group (146.2 19.3 months, p < 0.01). Both the control group (19 girls) and the case group (14 girls) had the same proportion of ancestors with precocious puberty (p = 0.90). There was no difference between the two groups in terms of African descent, socioeconomic strata and nutritional intakes (p > 0.05). The GnRH stimulated gonadotropin level test was performed in 20 patients with precocious puberty. Figs. 1, 2 and 3 show the PCR-RFLP reactions to the CYP1A1, CYP17, and CYP1B1 genes from the study participants. Table 3 displays the genotyping results. There was no difference in the distribution of the CYP1A1 Mspl (p = 0.86) and CYP17 genotypes (p = 0.12) between the two groups. The C/C mutant genotype from CYP1B1 Eco571 was more frequent in the Control group than in the Case group (p = 0.03). The odds ratios was 2.55 [95% CI—1.24–5.24] of the comparisons. Comments Investigation of the gene mechanisms possibly involved in early onset puberty might help to determine genetic markers for an earlier diagnosis and to develop new (pharmacogenetic) therapies with improvement in prognosis. However, little is still known about the genetic changes underlying sexual development at an early age [1]. Most studies assessing the influence of genetic factors on precocious puberty timing correlate central factors, which are associated with neurotransmitters [11,12]. Few studies have sought to evaluate a peripheral component as an early onset trigger [3,13]. Many researchers debate the role of estrogen in
Table 1 Oligonucleotides for PCR reactions. Gene
Initiators
Polymorphism/Enzyme
Reference
CYP1B1
S—50 -GTGGTTTTTGTCAACCAGTGG-30 AS—50 -GCCTCTTGCTTCTTATTGGCA-30 S—50 -TAGGAGTCTTGTCTCATGCCT-30 AS—50 -CAGTGAAGAGGTGTAGCCGCT-30 S—50 -CATTCGCACTCTGGAGTC–30 AS—50 -AGGCTCTTGGGGTACTTG–30
Transition Val/Leu codon 432/Eco 571
Zheng et al. [8]
Transition TA/CG region 30 UTR/MspI
Huang et al. [10]
Additional promotor SP1 (CCACC box)/Msp1A
Feigelson et al. [9]
CYP1A1 CYP17 S–Sense initiator. AS—Antisense initiator.
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Table 2 Clinical data from patients participating in the study. Control group (n = 104) Age at puberty onset (months) Menarche(months) Mother’s menarche (months) Ancestor* (number) Ethnicity of African descent Not of African descent *
Case group (n = 73)
p
114.9 19.3
75.8 14.9
<0.01
143.9 15.5 147.9 17.7
128.7 31.6 146.2 19.3
<0.01 0.88
19 77
14 56
26
16
0.90 0.87
Mother with precocious puberty.
gynecological disorders, such as endometriosis, leiomyoma, breast cancer, and precocious puberty [14]. In the latter, estradiol is believed to be not only a consequence but possibly a contributory factor in the early development of the hypothalamic–hypophyseal–ovarian axis as well [3–5]. This is the reason we chose polymorphisms which might modify steroidogenesis and thus serum estrogen levels and/or increase the metabolites with greater action on the steroid receptor. To date there are no reports on patients with precocious puberty who have been evaluated in relation to the polymorphisms of the CYP1A1, CYP17, and CYP1B1 genes. There are, however, studies that show the correlation between the polymorphisms of these genes and other dysfunctions such as endometriosis [7] and breast cancer [8,10,15]. A risk factor associated with breast cancer is known to be early menarche. As shown above, the enzymes of the CYP family might be related to precocious puberty due to their interference in estrogen metabolism.
The enzymes of the CYP family which participate in steroidogenesis are as important in estrogen production as they are in estrogen bioavailability and degradation. Several studies have identified mutations in the enzyme complex that may be influential in the onset and prognosis of diseases [16]. This is why we chose to study the CYP1A1, CYP17, and CYP1B1 genes. The CYP1A1 gene participates in estrogen hydroxylation activity generating its hydroxy estradiol metabolites, (HE2), 4-HE2, and 16a-HE2, which have very potent hormone action [10] on the estrogen receptor. Gorai et al. [3] studied 317 postmenopausal Japanese women and concluded there was no significant difference in terms of CYP1A1 mutation and age at menarche between the study groups. Nor did we observe a higher frequency of such polymorphism in the CYP1A1 gene in the precocious puberty patients in our study. The CYP17 is the enzyme that catalyzes 17a-hydroxylase and 17–20-lyase activity, increasing androgen and estrogen biosynthesis [17]. In studies examining the CYP17 polymorphism in women with breast cancer, Feigelson et al. [15] detected the A2 allele in women of a younger age at menarche, while Haiman et al. [18] found no significant difference. Our data suggest the gene does not participate in the timing of precocious puberty. The CYP1B1 gene is located on chromosome 2, region 2p21–22, contains three exons, and codes the P450-1B1 cytochrome, the enzyme responsible for the oxidative metabolism of estradiol. Its polymorphism results from the replacement of a guanine base (G) with a cytosine (C) in exon 3, which triggers the substitution of leucine for valine in codon 432, creating an Eco 571 restriction site. The CYP1B1 gene plays an important role in the hydroxylation and conjugation processes catalyzing the formation of 4-HE2 [8]. CYP1B1 expression is regulated by estradiol and it binds to its receptor. Changes at the CYP1B1 expression level not only modify the intensity of estrogen action, but may also modify the profile of its physiological effect on the liver and target tissues.
Fig. 1. Representative gel of the polymorphism pattern obtained from Mspl restriction of enlarged samples for CYP1A1. P, standard molecular weight of 100 base pairs (bp). Samples with only one band indicate a wild profile, (340 bp, e.g., lanes 12–14), while samples with two (210 and 130 bp, e.g., lane 37) and three bands (340, 210 e 130 bp, e.g., lanes 4–5) indicate the homozygous and heterozygous profile for mutation. The arrows point at migration of the molecular weight standard in 500 bp.
C.N. Matsuzaki et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 140–144
143
Fig. 2. Representative gel of the polymorphism pattern obtained from Msp1A restriction of enlarged samples for CYP17. P, standard molecular weight of 100 base pairs. Samples with only one band indicate a wild profile, (A1/A1, e.g. lanes 13–17), while samples with three (A1/A2, e.g., lanes 26–39) indicate the heterozygous profile. The arrows point at the approximate migration height of the three fragments that were obtained.
Fig. 3. Representative gel of the polymorphism pattern obtained from Eco 571 restriction of enlarged samples for CYP1B1. P, standard molecular weight of 100 base pairs. Samples with only one band indicate a wild profile, (G/G, e.g. lanes 19–21), while samples with three (A1/A2, e.g., lanes 26–39) and two bands (C/C, e.g., lanes 61–64) indicate the heterozygous and the homozygous profile, respectively. The arrows point at the approximate migration height of the three fragments that were obtained.
In target tissues such as the myometrium, in leiomyomas, and in benign breast tumors, an increase in 4-HE2 has been detected. At these localities, a rise in CYP1B1 expression level has also been confirmed, and the predominant route is 4-hydroxylation of
estrogen. Besides, the CYP1B1 expression level is abundant in tumor tissues, more so than in normal tissues [19]. It appears that the association between CYP1B1 and mammary and endometrial carcinogenesis is formed primarily via generation
Table 3 Results from genotyping. Genotyping
CYP1A1 (n) CYP17 (n) CYP1B1 (n)
Control group (n = 104)
Case group (n = 73)
p
Wild
Heterozygote
Homozygote
Wild
Heterozygote
Homozygote
60(W/W) 35(A1/A) 29 (G/G)
41 (W/VT) 69 (A1/A2) 38 (G/C)
3 (VT/VT) 0 (A2/A2) 37(C/C)
40(W/W) 33(A1/A) 23 (G/G)
30 (W/VT) 40 (A1/A2) 37 (G/C)
3 (VT/VT) 0 (A2/A2) 13 (C/C)
0.86 0.12 0.03
144
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of toxic metabolites, the so-called pro-carcinogens and promutagens, rather than hormone potency, given that 4-hydroxylation of estrogen by CYP1B1 leads to a reduction in estrogen activity [19]. It should be pointed out that increased SHBG production in the liver may diminish the availability not only of the metabolite but of estrogen itself [20]. Another hypothesis is the non-participation of this polymorphism in pubertal development. In fact, Mitchell et al. [21] studied 152 white women and concluded that the CYP1B1 polymorphism was not associated with age at menarche. Finally, we found an inverse relationship between CYP1B1 and precocious puberty. Our data suggest the CYP1B1 Eco571 gene variant is associated with puberty timing. Hence, further research should focus on the functional investigation of this particular polymorphism. Condensation The CYP1B1 Eco571 gene variant may be associated with puberty timing in patients with precocious puberty. Acknowledgement We would like to thank Prof Maria Lucia Flynn for manuscript revision. References [1] Jarzabek-Bielecka G, Warchoł-Biedermann K, Sowin´ska E, Wachowiak-Ochman´ska K. Precocious puberty. Ginekol Pol 2011;82(4):281–6. [2] Teles MG, Bianco SD, Brito VN, et al. A GPR54-activating mutation in a patient with central precocious puberty. N Engl J Med 2008;358(7):709–15. [3] Gorai I, Tanaka K, Inada M, et al. Estrogen-metabolizing gene polymorphisms, but not estrogen receptor-alpha gene polymorphisms, are associated with the onset of menarche in healthy postmenopausal Japanese women. J Clin Endocrinol Metab 2003;88(2):799–803. [4] Moenter SM, Defazio RA, Straume M, Nunemaker CS. Steroid regulation of GnRH neurons. Ann NY Acad Sci 2003;1007:143–52. [5] Ronnekleiv OK, Bosch MA, Zhang C. 17b-oestradiol regulation of gonadotrophin-releasing hormone neuronal excitability. J Neuroendocrinol 2012;24(1): 122–30.
[6] Parent AS, Rasier G, Gerard A, et al. Early onset of puberty: tracking genetic and environmental factors. Horm Res 2005;64(2):41–7. [7] Huber A, Keck CC, Hefler LA, et al. Ten estrogen-related polymorphisms and endometriosis: a study of multiple gene–gene interactions. Obstet Gynecol 2005;106(5 Pt 1):1025–31. [8] Zheng W, Xie DW, Jin F, et al. Genetic polymorphism of cytochrome P450-1B1 and risk of breast cancer. Cancer Epidemiol Biomarkers Prev 2000;9(2):147– 50. [9] Feigelson HS, McKean-Cowdin R, Pike MC, et al. Cytochrome P450c17alpha gene (CYP17) polymorphism predicts use of hormone replacement therapy. Cancer Res 1999;59(16):3908–10. [10] Huang CS, Chern HD, Chang KJ, Cheng CW, Hsu SM, Shen CY. Breast cancer risk associated with genotype polymorphism of the estrogen-metabolizing genes CYP17, CYP1A1, and COMT: a multigenic study on cancer susceptibility. Cancer Res 1999;59(19):4870–5. [11] Holanda FS, Tufik S, Bignotto M, et al. Evaluation of melatonin on the precocious puberty: a pilot study. Gynecol Endocrinol 2011;27(8):519–23. [12] Navarro VM, Castellano JM, Garcı´a-Galiano D, Tena-Sempere M. Neuroendocrine factors in the initiation of puberty: the emergent role of kisspeptin. Rev Endocr Metab Disord 2007;8:11–20. [13] Rasier G, Matagne V, Parent AS, Gerard A, Lebrethon MC, Bourguignon JP. Estradiol and dichlorodiphenyltrichloroethane (DDT) administration in infantile female rats: similar stimulation of gonadotropin-releasing hormone (GnRH) secretion in vitro and sexual maturation in vivo through different receptor mechanisms. In: Proceedings of the 87th annual meeting of the endocrine society. San Diego, CA: Endocrine Society; 2005. p. 190. [14] Paterni I, Granchi C, Katzenellenbogen JA, Minutolo F. Estrogen receptors alpha (ERa) and beta (ERb): subtype-selective ligands and clinical potential. Steroids 2014. http://dx.doi.org/10.1016/j.steroids.2014.06.012. pii: S0039128X(14)00151-2 [Epub ahead of print]. [15] Feigelson HS, Coetzee GA, Kolonel LN, Ross RK, Henderson BE. A polymorphism in the CYP17 gene increases the risk of breast cancer. Cancer Res 1997;57(6):1063–5. [16] van Schaik RH. Cancer treatment and pharmacogenetics of cytochrome P450 enzymes. Invest New Drugs 2005;23(6):513–22. [17] Nogueira Jr RC, Costa AM, Silva ID, et al. Influence of the CYP17 polymorphism on vasomotor symptoms in postmenopausal women: a pilot study. Climacteric 2011;14(5):537–43. [18] Haiman CA, Hankinson SE, Spiegelman D, et al. The relationship between a polymorphism in CYP17 with plasma hormone levels and breast cancer. Cancer Res 1999;59(5):1015–20. [19] Tsuchiya Y, Nakajima M, Yokoi T. Cytochrome P450-mediated metabolism of estrogens and its regulation in human. Cancer Lett 2005;227(2):115–24. [20] Zimmerman Y, Eijkemans MJ, Coelingh Bennink HJ, Blankenstein MA, Fauser BC. The effect of combined oral contraception on testosterone levels in healthy women: a systematic review and meta-analysis. Hum Reprod Update 2014;20(1):76–105. [21] Mitchell ES, Farin FM, Stapleton PL, et al. Association of estrogen-related polymorphisms with age at menarche, age at final menstrual period, and stages of the menopausal transition. Menopause 2008;15(1):105–11.
Contents lists available at ScienceDirect
European Journal of Obstetrics & Gynecology and Reproductive Biology journal homepage: www.elsevier.com/locate/ejogrb
Are CYP1A1, CYP17 and CYP1B1 mutation genes involved on girls with precocious puberty? A pilot study Cezar Noboru Matsuzaki a, Jose´ Maria Soares Ju´nior a,*, Durval Damiani b, Raymundo Soares de Azevedo Neto c, Ka´tia Caˆndido Carvalho a, Felisbela Soares de Holanda a, Eline Maria Stafuzza a, Jose´ Alcione Macedo Almeida a, Edmund Chada Baracat a a
Disciplina de Ginecologia do Departamento de Obstetrı´cia e Ginecologia da Faculdade de Medicina da Universidade de Sa˜o Paulo, 05403000, Sa˜o Paulo, SP, Brazil b Departamento de Pediatria da Faculdade de Medicina da Universidade de Sa˜o Paulo, 05403000, Sa˜o Paulo, SP, Brazil c Departamento de Patologia da Faculdade de Medicina da Universidade de Sa˜o Paulo, 05403000, Sa˜o Paulo, SP, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 24 June 2014 Received in revised form 23 July 2014 Accepted 29 July 2014
Objectives: To investigate three genes associated with puberty timing in girls with central precocious puberty by evaluating the association between polymorphism in the gene sequence codifying the enzymes participating in steroidogenesis, CYP1A1, CYP17, and CYP1B1 and central precocious puberty. Study design: A total of 177 patients was included and divided into two groups: Case group with 73 girls diagnosed with central precocious puberty; Control group with 104 girls with puberty onset after 8 years of age who were followed at the Sector of Gynecology of Childhood and Adolescence, Division of Gynecology Clinic, HC-FMUSP. Polymorphism presence was assessed in the genes involved in estrogen metabolism (CYP1A1, CYP17, and CYP1B1) by the restriction fragment length polymorphism (RFLP) technique using DNA from peripheral blood. Results: No significant difference in the distribution of the CYP1A1 Mspl (p = 0.86) and CYP17 (p = 0.12) genotypes was detected between the two study groups. As for CYP1B1 Eco571, the mutated C/C genotype was found to be more frequent in the control group than in the case group (p = 0.03). Conclusion: Our data suggest the CYP1B1 Eco571 gene variant is associated with puberty timing. ß 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: Genetic polymorphism Estrogens Precocious puberty
Introduction Puberty timing is still unknown. Its mechanism is the source of debate involving genetic susceptibility along with nutritional and environmental factors. The genetic factors influencing puberty timing are yet to be determined [1]. Most studies on the timing of early puberty correlate central factors, which are associated with neurotransmitters, such as glutamate and the kisspeptin/GPR54 complex [2]. There are few studies assessing the peripheral factors which may influence early activation of the hypophyseal–hypothalamic axis. For some researchers, estradiol would be not only the consequence of puberty onset but also a contributory factor in the development of the hypophyseal–hypothalamic axis [3–5]. In a few of the prospective genes, such as those codifying the CYP enzyme complex,
which control the biosynthesis, action, and metabolism of the sex steroids, polymorphisms have been found associated with variations on puberty timing [6]. Such genes are also associated with other gynecological dysfunctions involving estrogen action, such as leiomyoma, endometriosis, and breast cancer [7,8]. The CYP17 gene is believed to be involved in increased production of sex steroids [9]. On the other hand, the CYPA1 and CYPB1 polymorphisms may be related to more active metabolites (4-OH estradiol and 16-OH estradiol) acting on the estrogen receptor and on estrogen-signaling pathways [10]. Therefore, our study aimed at investigating the possibility that the association between such polymorphisms and the central precocious puberty process is brought on by increased estrogen action and attendant GnRH release.
Materials and methods * Corresponding author. Tel.: +55 11 26617838; fax: +55 11 26617838. E-mail address: [email protected] (J.M.S. Ju´nior). http://dx.doi.org/10.1016/j.ejogrb.2014.07.042 0301-2115/ß 2014 Elsevier Ireland Ltd. All rights reserved.
A total of 177 patients was selected from a group of 1685 patients being treated at the Sector of Gynecology of Childhood and
C.N. Matsuzaki et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 181 (2014) 140–144
Adolescence, Gynecology Discipline, Department of Obstetrics and Gynecology, School of Medicine of the University of Sa˜o Paulo (FMUSP). The 177 patients were allocated to two groups: Case group with 73 girls with a current or previous diagnosis of precocious puberty and Control group with 104 girls who showed no signs of puberty until they were 8 years old. The diagnosis of central precocious puberty was based on the clinical features, laboratory exams (LH, FSH, GnRH stimulated gonadotropin levels, estradiol levels), bone age, pelvic ultrasound and brain MRI. The exclusion criteria comprised presence of ovarian or adrenal neoplasms, Cushing syndrome, McCune–Albright syndrome, eye disorders, clinical or laboratory hypothyroidism, use of hormone medications prior to diagnosis, and use of psychotropic or antiepileptic medication. The project was approved by the Ethics Committee for Analysis of Research Projects at HC-FMUSP and FMUSP (CAPPesq–research protocol No. 1120/09). The volunteers were included after their legal guardians freely signed an informed consent statement. Polymorphonuclear leukocytes separated from blood samples were utilized for the patients’ total DNA extraction, carried out with the QIAmp1 DNA mini Blood (Qiagen) kit. The CYP1A1 polymorphism derives from the replacement of cytosine with thymine in the 30 noncoding region, which creates an Mspl restriction site allowing the identification of the following genotypes: CYP1A1wt/wt (wild homozygote), CYP1A1wt/vt (heterozygote), and CYP1A1vt/vt (mutant homozygote) [10]. The 50 region, untranslated from CYP17, becomes polymorphic with the substitution of thymine for cytosine, creating an Mspl restriction site, which identifies two alleles, A1 (wild) and 2 (mutant). The latter increases CYP17 expression, boosting estradiol biosynthesis [9]. Replacing a guanine base with cytosine in exon 3 of the CYP1B1 gene leads to the substitution of leucine for valine in codon 432, creating an Eco 571 restriction site, enabling identification of the G/ G (wild homozygote), G/C (heterozygote), and C/C (mutant homozygote) genotypes [8]. Analysis of the polymorphisms in the CYP1A1, CYP17, and CYP1B1 genes was performed using the PCR-RFLP (restriction fragment length polymorphism) technique. The PCR reactions were produced with 50 ng of total DNA in 20 mM Tris–HCl (pH 8.4), 50 mM KCl, 0.2 mM dNTP, 0.1 mM of each initiator, 1.5 mM of MgSO2, and 0.4 U of Platinum Taq DNA Polymerase High Fidelity (Life Biosciences). The cycling conditions were as follows: preheating to 95 8C for 10 min, followed by 40 cycles at 95 8C for 15 s, at 55 8C for 1 min, at 72 8C for 1 min, followed by a final extension step at 70 8C for 7 min. A specific initiator oligonucleotide was used for each of the polymorphisms that were being investigated. The oligonucleotides are described in Table 1. Following amplification, the PCR fragments corresponding to the CYP1A1, CYP17, and CYP1B1 genes were digested with the restriction enzymes (Promega, Brazil) specific for each polymorphism as reported in the literature (Table 1). Fragments were analyzed in 1% to 3% agarose gel and stained with ethidium
141
bromide. The PCR and RFLP conditions were those already standardized for each gene as described in the literature [8–10]. After data collection, the mean, median, and standard deviation were calculated. Next, sample homogeneity was assessed by the Bartlett test. Results were displayed as mean standard error of the mean. When the two groups were involved in a comparison, the chisquare test was employed. For proportion analysis, the Fisher test was used, and for polymorphisms, a test of Hardy–Weinberg equilibrium. Analyses were carried out with the SPSS version 16.0 software program. In all tests, the significance level for rejecting the null hypothesis was set at 5% or 0.05 (p < 0.05). Results The clinical and epidemiological characteristics of the 73 girls with central precocious puberty (Case group) and of the 104 girls with normal puberty (Control group) making up our study sample are shown in Table 2. At the onset of puberty, the mean age of the girls in the Control group showed they were older (114.9 19.3 months) than those in the Case group (75.8 14.9 months, p < 0.01). Also, at menarche, the mean age of the control group (143.9 15.5 months) was significantly greater than that of the case group (128.7 31.6 months). There was no difference in the mother’s age at menarche between the two groups, i.e., the Control group (147.9 17.7 months) and the Case group (146.2 19.3 months, p < 0.01). Both the control group (19 girls) and the case group (14 girls) had the same proportion of ancestors with precocious puberty (p = 0.90). There was no difference between the two groups in terms of African descent, socioeconomic strata and nutritional intakes (p > 0.05). The GnRH stimulated gonadotropin level test was performed in 20 patients with precocious puberty. Figs. 1, 2 and 3 show the PCR-RFLP reactions to the CYP1A1, CYP17, and CYP1B1 genes from the study participants. Table 3 displays the genotyping results. There was no difference in the distribution of the CYP1A1 Mspl (p = 0.86) and CYP17 genotypes (p = 0.12) between the two groups. The C/C mutant genotype from CYP1B1 Eco571 was more frequent in the Control group than in the Case group (p = 0.03). The odds ratios was 2.55 [95% CI—1.24–5.24] of the comparisons. Comments Investigation of the gene mechanisms possibly involved in early onset puberty might help to determine genetic markers for an earlier diagnosis and to develop new (pharmacogenetic) therapies with improvement in prognosis. However, little is still known about the genetic changes underlying sexual development at an early age [1]. Most studies assessing the influence of genetic factors on precocious puberty timing correlate central factors, which are associated with neurotransmitters [11,12]. Few studies have sought to evaluate a peripheral component as an early onset trigger [3,13]. Many researchers debate the role of estrogen in
Table 1 Oligonucleotides for PCR reactions. Gene
Initiators
Polymorphism/Enzyme
Reference
CYP1B1
S—50 -GTGGTTTTTGTCAACCAGTGG-30 AS—50 -GCCTCTTGCTTCTTATTGGCA-30 S—50 -TAGGAGTCTTGTCTCATGCCT-30 AS—50 -CAGTGAAGAGGTGTAGCCGCT-30 S—50 -CATTCGCACTCTGGAGTC–30 AS—50 -AGGCTCTTGGGGTACTTG–30
Transition Val/Leu codon 432/Eco 571
Zheng et al. [8]
Transition TA/CG region 30 UTR/MspI
Huang et al. [10]
Additional promotor SP1 (CCACC box)/Msp1A
Feigelson et al. [9]
CYP1A1 CYP17 S–Sense initiator. AS—Antisense initiator.
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Table 2 Clinical data from patients participating in the study. Control group (n = 104) Age at puberty onset (months) Menarche(months) Mother’s menarche (months) Ancestor* (number) Ethnicity of African descent Not of African descent *
Case group (n = 73)
p
114.9 19.3
75.8 14.9
<0.01
143.9 15.5 147.9 17.7
128.7 31.6 146.2 19.3
<0.01 0.88
19 77
14 56
26
16
0.90 0.87
Mother with precocious puberty.
gynecological disorders, such as endometriosis, leiomyoma, breast cancer, and precocious puberty [14]. In the latter, estradiol is believed to be not only a consequence but possibly a contributory factor in the early development of the hypothalamic–hypophyseal–ovarian axis as well [3–5]. This is the reason we chose polymorphisms which might modify steroidogenesis and thus serum estrogen levels and/or increase the metabolites with greater action on the steroid receptor. To date there are no reports on patients with precocious puberty who have been evaluated in relation to the polymorphisms of the CYP1A1, CYP17, and CYP1B1 genes. There are, however, studies that show the correlation between the polymorphisms of these genes and other dysfunctions such as endometriosis [7] and breast cancer [8,10,15]. A risk factor associated with breast cancer is known to be early menarche. As shown above, the enzymes of the CYP family might be related to precocious puberty due to their interference in estrogen metabolism.
The enzymes of the CYP family which participate in steroidogenesis are as important in estrogen production as they are in estrogen bioavailability and degradation. Several studies have identified mutations in the enzyme complex that may be influential in the onset and prognosis of diseases [16]. This is why we chose to study the CYP1A1, CYP17, and CYP1B1 genes. The CYP1A1 gene participates in estrogen hydroxylation activity generating its hydroxy estradiol metabolites, (HE2), 4-HE2, and 16a-HE2, which have very potent hormone action [10] on the estrogen receptor. Gorai et al. [3] studied 317 postmenopausal Japanese women and concluded there was no significant difference in terms of CYP1A1 mutation and age at menarche between the study groups. Nor did we observe a higher frequency of such polymorphism in the CYP1A1 gene in the precocious puberty patients in our study. The CYP17 is the enzyme that catalyzes 17a-hydroxylase and 17–20-lyase activity, increasing androgen and estrogen biosynthesis [17]. In studies examining the CYP17 polymorphism in women with breast cancer, Feigelson et al. [15] detected the A2 allele in women of a younger age at menarche, while Haiman et al. [18] found no significant difference. Our data suggest the gene does not participate in the timing of precocious puberty. The CYP1B1 gene is located on chromosome 2, region 2p21–22, contains three exons, and codes the P450-1B1 cytochrome, the enzyme responsible for the oxidative metabolism of estradiol. Its polymorphism results from the replacement of a guanine base (G) with a cytosine (C) in exon 3, which triggers the substitution of leucine for valine in codon 432, creating an Eco 571 restriction site. The CYP1B1 gene plays an important role in the hydroxylation and conjugation processes catalyzing the formation of 4-HE2 [8]. CYP1B1 expression is regulated by estradiol and it binds to its receptor. Changes at the CYP1B1 expression level not only modify the intensity of estrogen action, but may also modify the profile of its physiological effect on the liver and target tissues.
Fig. 1. Representative gel of the polymorphism pattern obtained from Mspl restriction of enlarged samples for CYP1A1. P, standard molecular weight of 100 base pairs (bp). Samples with only one band indicate a wild profile, (340 bp, e.g., lanes 12–14), while samples with two (210 and 130 bp, e.g., lane 37) and three bands (340, 210 e 130 bp, e.g., lanes 4–5) indicate the homozygous and heterozygous profile for mutation. The arrows point at migration of the molecular weight standard in 500 bp.
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Fig. 2. Representative gel of the polymorphism pattern obtained from Msp1A restriction of enlarged samples for CYP17. P, standard molecular weight of 100 base pairs. Samples with only one band indicate a wild profile, (A1/A1, e.g. lanes 13–17), while samples with three (A1/A2, e.g., lanes 26–39) indicate the heterozygous profile. The arrows point at the approximate migration height of the three fragments that were obtained.
Fig. 3. Representative gel of the polymorphism pattern obtained from Eco 571 restriction of enlarged samples for CYP1B1. P, standard molecular weight of 100 base pairs. Samples with only one band indicate a wild profile, (G/G, e.g. lanes 19–21), while samples with three (A1/A2, e.g., lanes 26–39) and two bands (C/C, e.g., lanes 61–64) indicate the heterozygous and the homozygous profile, respectively. The arrows point at the approximate migration height of the three fragments that were obtained.
In target tissues such as the myometrium, in leiomyomas, and in benign breast tumors, an increase in 4-HE2 has been detected. At these localities, a rise in CYP1B1 expression level has also been confirmed, and the predominant route is 4-hydroxylation of
estrogen. Besides, the CYP1B1 expression level is abundant in tumor tissues, more so than in normal tissues [19]. It appears that the association between CYP1B1 and mammary and endometrial carcinogenesis is formed primarily via generation
Table 3 Results from genotyping. Genotyping
CYP1A1 (n) CYP17 (n) CYP1B1 (n)
Control group (n = 104)
Case group (n = 73)
p
Wild
Heterozygote
Homozygote
Wild
Heterozygote
Homozygote
60(W/W) 35(A1/A) 29 (G/G)
41 (W/VT) 69 (A1/A2) 38 (G/C)
3 (VT/VT) 0 (A2/A2) 37(C/C)
40(W/W) 33(A1/A) 23 (G/G)
30 (W/VT) 40 (A1/A2) 37 (G/C)
3 (VT/VT) 0 (A2/A2) 13 (C/C)
0.86 0.12 0.03
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of toxic metabolites, the so-called pro-carcinogens and promutagens, rather than hormone potency, given that 4-hydroxylation of estrogen by CYP1B1 leads to a reduction in estrogen activity [19]. It should be pointed out that increased SHBG production in the liver may diminish the availability not only of the metabolite but of estrogen itself [20]. Another hypothesis is the non-participation of this polymorphism in pubertal development. In fact, Mitchell et al. [21] studied 152 white women and concluded that the CYP1B1 polymorphism was not associated with age at menarche. Finally, we found an inverse relationship between CYP1B1 and precocious puberty. Our data suggest the CYP1B1 Eco571 gene variant is associated with puberty timing. Hence, further research should focus on the functional investigation of this particular polymorphism. Condensation The CYP1B1 Eco571 gene variant may be associated with puberty timing in patients with precocious puberty. Acknowledgement We would like to thank Prof Maria Lucia Flynn for manuscript revision. References [1] Jarzabek-Bielecka G, Warchoł-Biedermann K, Sowin´ska E, Wachowiak-Ochman´ska K. Precocious puberty. Ginekol Pol 2011;82(4):281–6. [2] Teles MG, Bianco SD, Brito VN, et al. A GPR54-activating mutation in a patient with central precocious puberty. N Engl J Med 2008;358(7):709–15. [3] Gorai I, Tanaka K, Inada M, et al. Estrogen-metabolizing gene polymorphisms, but not estrogen receptor-alpha gene polymorphisms, are associated with the onset of menarche in healthy postmenopausal Japanese women. J Clin Endocrinol Metab 2003;88(2):799–803. [4] Moenter SM, Defazio RA, Straume M, Nunemaker CS. Steroid regulation of GnRH neurons. Ann NY Acad Sci 2003;1007:143–52. [5] Ronnekleiv OK, Bosch MA, Zhang C. 17b-oestradiol regulation of gonadotrophin-releasing hormone neuronal excitability. J Neuroendocrinol 2012;24(1): 122–30.
[6] Parent AS, Rasier G, Gerard A, et al. Early onset of puberty: tracking genetic and environmental factors. Horm Res 2005;64(2):41–7. [7] Huber A, Keck CC, Hefler LA, et al. Ten estrogen-related polymorphisms and endometriosis: a study of multiple gene–gene interactions. Obstet Gynecol 2005;106(5 Pt 1):1025–31. [8] Zheng W, Xie DW, Jin F, et al. Genetic polymorphism of cytochrome P450-1B1 and risk of breast cancer. Cancer Epidemiol Biomarkers Prev 2000;9(2):147– 50. [9] Feigelson HS, McKean-Cowdin R, Pike MC, et al. Cytochrome P450c17alpha gene (CYP17) polymorphism predicts use of hormone replacement therapy. Cancer Res 1999;59(16):3908–10. [10] Huang CS, Chern HD, Chang KJ, Cheng CW, Hsu SM, Shen CY. Breast cancer risk associated with genotype polymorphism of the estrogen-metabolizing genes CYP17, CYP1A1, and COMT: a multigenic study on cancer susceptibility. Cancer Res 1999;59(19):4870–5. [11] Holanda FS, Tufik S, Bignotto M, et al. Evaluation of melatonin on the precocious puberty: a pilot study. Gynecol Endocrinol 2011;27(8):519–23. [12] Navarro VM, Castellano JM, Garcı´a-Galiano D, Tena-Sempere M. Neuroendocrine factors in the initiation of puberty: the emergent role of kisspeptin. Rev Endocr Metab Disord 2007;8:11–20. [13] Rasier G, Matagne V, Parent AS, Gerard A, Lebrethon MC, Bourguignon JP. Estradiol and dichlorodiphenyltrichloroethane (DDT) administration in infantile female rats: similar stimulation of gonadotropin-releasing hormone (GnRH) secretion in vitro and sexual maturation in vivo through different receptor mechanisms. In: Proceedings of the 87th annual meeting of the endocrine society. San Diego, CA: Endocrine Society; 2005. p. 190. [14] Paterni I, Granchi C, Katzenellenbogen JA, Minutolo F. Estrogen receptors alpha (ERa) and beta (ERb): subtype-selective ligands and clinical potential. Steroids 2014. http://dx.doi.org/10.1016/j.steroids.2014.06.012. pii: S0039128X(14)00151-2 [Epub ahead of print]. [15] Feigelson HS, Coetzee GA, Kolonel LN, Ross RK, Henderson BE. A polymorphism in the CYP17 gene increases the risk of breast cancer. Cancer Res 1997;57(6):1063–5. [16] van Schaik RH. Cancer treatment and pharmacogenetics of cytochrome P450 enzymes. Invest New Drugs 2005;23(6):513–22. [17] Nogueira Jr RC, Costa AM, Silva ID, et al. Influence of the CYP17 polymorphism on vasomotor symptoms in postmenopausal women: a pilot study. Climacteric 2011;14(5):537–43. [18] Haiman CA, Hankinson SE, Spiegelman D, et al. The relationship between a polymorphism in CYP17 with plasma hormone levels and breast cancer. Cancer Res 1999;59(5):1015–20. [19] Tsuchiya Y, Nakajima M, Yokoi T. Cytochrome P450-mediated metabolism of estrogens and its regulation in human. Cancer Lett 2005;227(2):115–24. [20] Zimmerman Y, Eijkemans MJ, Coelingh Bennink HJ, Blankenstein MA, Fauser BC. The effect of combined oral contraception on testosterone levels in healthy women: a systematic review and meta-analysis. Hum Reprod Update 2014;20(1):76–105. [21] Mitchell ES, Farin FM, Stapleton PL, et al. Association of estrogen-related polymorphisms with age at menarche, age at final menstrual period, and stages of the menopausal transition. Menopause 2008;15(1):105–11.